Normalization of data

ABSTRACT

Methods for normalizing output from an instrument employing a reference standard or non-fluorescing substance disposed within at least one of a plurality of reaction chambers. The method comprises collecting and analyzing a signal associated with the reference standard or non-fluorescing substance to determine a normalizing bias. The normalizing bias is then applied to the data signal collected from a remainder of the plurality of reaction chambers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of patent application Ser. No.12/176,656 filed Jul. 21, 2008, which is a continuation of patentapplication Ser. No. 11/087,103 filed Mar. 22, 2005, now U.S. Pat. No.7,417,726, which is a continuation-in-part of patent application Ser.No. 10/913,601 filed Aug. 5, 2004, now U.S. Pat. No. 7,233,393. Patentapplication Ser. No. 10/944,673 filed Sep. 17, 2004, now abandoned, andpatent application Ser. No. 10/944,691 filed Sep. 17, 2004, nowabandoned. Patent application Ser. No. 10/944,673 claims a benefit toU.S. Provisional Application No. 60/504,500 filed Sep. 19, 2003; U.S.Provisional Application No. 60/504,052 filed Sep. 19, 2003; U.S.Provisional Application No. 60/589,224 filed Jul. 19, 2004; U.S.Provisional Application No. 60/589,225 filed Jul. 19, 2004; and U.S.Provisional Application No. 60/601,716 filed Aug. 13, 2004. Patentapplication Ser. No. 10/944,691 is a continuation-in-part of patentapplication Ser. No. 10/913,601 filed Aug. 5, 2004, which further claimsthe benefit of U.S. Provisional Application No. 60/504,052 filed Sep.19, 2003; U.S. Provisional Application No. 60/504,500 filed Sep. 19,2003; U.S. Provisional Application No. 60/589,224 filed Jul. 19, 2004;U.S. Provisional Application No. 60/589,225 filed Jul. 19, 2004; andU.S. Provisional Application No. 60/601,716 filed Aug. 13, 2004. All ofwhich are incorporated here by reference.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages, regardless of the format ofsuch literature and similar materials, are expressly incorporated byreference in their entirety for any purpose. In the event that one ormore of the incorporated literature and similar materials differs fromor contradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

INTRODUCTION

Currently, genomic analysis, including that of the estimated 30,000human genes is a major focus of basic and applied biochemical andpharmaceutical research. Such analysis may aid in developingdiagnostics, medicines, and therapies for a wide variety of disorders.However, the complexity of the human genome and the interrelatedfunctions of genes often make this task difficult. There is a continuingneed for methods and apparatus to aid in such analysis.

DRAWINGS

The skilled artisan will understand that the drawings, described herein,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1( a) is a perspective view illustrating a high-density sequencedetection system according to some embodiments of the present teachings;

FIG. 1( b) is a perspective view illustrating a high-density sequencedetection system according to some embodiments of the present teachings;

FIG. 1( c) is a side view illustrating the high-density sequencedetection system of FIG. 1( b);

FIG. 2 is a top perspective view illustrating a microplate in accordancewith some embodiments;

FIG. 3 is a top perspective view illustrating a microplate in accordancewith some embodiments;

FIG. 4 is an enlarged perspective view illustrating a microplate inaccordance with some embodiments comprising a plurality of wellscomprising a circular rim portion;

FIG. 5 is an enlarged perspective view illustrating a microplate inaccordance with some embodiments comprising a plurality of wellscomprising a square-shaped rim portion;

FIG. 6 is a cross-sectional view illustrating a well comprising apressure relief bore according to some embodiments;

FIG. 7 is a cross-sectional view illustrating the well of FIG. 6 whereinthe pressure relief bore is partially filled;

FIG. 8 is a cross-sectional view illustrating a well comprising anoffset pressure relief bore according to some embodiments, being filledby a spotting device;

FIG. 9 is a cross-sectional view illustrating the well of FIG. 8 beingfilled by a micro-piezo dispenser;

FIG. 10 is a cross-sectional view illustrating a microplate employing aplurality of apertures, a foil seal, and a sealing cover according tosome embodiments;

FIG. 11 is a top view illustrating a microplate in accordance with someembodiments comprising one or more grooves;

FIG. 12 is an enlarged top view illustrating a corner of the microplateillustrated in FIG. 11,

FIG. 13 is a cross-sectional view of the microplate of FIG. 12 takenalong Line 13-13;

FIG. 14 is an enlarged top view illustrating a corner of a microplateaccording to some embodiments;

FIG. 15 is a cross-sectional view of the microplate of FIG. 14 takenalong Line 15-15;

FIG. 16 is a top view illustrating a microplate in accordance with someembodiments comprising at least one thermally isolated portion;

FIG. 17 is a side view illustrating the microplate of FIG. 16;

FIG. 18 is a bottom view illustrating the microplate of FIG. 16;

FIG. 19 is an enlarged cross-sectional view illustrating the microplateof FIG. 16 taken along Line 19-19;

FIG. 20 is an exploded perspective view illustrating a filling apparatusaccording to some embodiments;

FIG. 21 is a cross-sectional perspective view of the filling apparatusof FIG. 20;

FIG. 22( a) is a cross-sectional perspective view of a filling apparatusaccording to some embodiments;

FIG. 22( b) is a cross-sectional view of a portion of a fillingapparatus comprising a plurality of staging capillaries, microfluidicchannels, and ramp features according to some embodiments;

FIG. 23( a) is a top schematic view of a filling apparatus according tosome embodiments;

FIG. 23( b) is a top perspective view of a portion of a fillingapparatus comprising a plurality of staging capillaries, microfluidicchannels, and ramp features according to some embodiments;

FIG. 24 is a bottom perspective view of an output layer of a fillingapparatus comprising spacer features according to some embodiments;

FIGS. 25( a)-(f) are top schematic views of a filling apparatusaccording to some embodiments;

FIG. 26 is a cross-sectional view illustrating a well of a microplateaccording to some embodiments;

FIG. 27 is a cross-sectional view illustrating a well of an invertedmicroplate according to some embodiments;

FIG. 28 is a cross-sectional view illustrating a sealing cover accordingto some embodiments;

FIG. 29 is a cross-sectional view illustrating a hot roller apparatusthat can be used to seal a sealing cover to a microplate according tosome embodiments;

FIG. 30 is a cross-sectional view illustrating a pressure clamp systemaccording to some embodiments comprising an inflatable transparent bag;

FIG. 31 is a cross-sectional view illustrating a pressure clamp systemaccording to some embodiments comprising a moveable transparent window;

FIG. 32 is a cross-sectional view illustrating a pressure clamp systemaccording to some embodiments comprising an inverted microplate;

FIG. 33 is a cross-sectional view illustrating a pressure clamp systemaccording to some embodiments comprising a plurality of apertures in amicroplate;

FIG. 34 is a cross-sectional view illustrating a pressure clamp systemaccording to some embodiments comprising a pressure chamber engaging asealing cover;

FIG. 35 is a cross-sectional view illustrating a pressure clamp systemaccording to some embodiments comprising a pressure chamber usedtogether with an inverted microplate;

FIG. 36 is a cross-sectional view illustrating a pressure clamp systemaccording to some embodiments comprising a pressure chamber usedtogether with a microplate comprising a plurality of apertures;

FIG. 37 is a cross-sectional view illustrating a pressure clamp systemaccording to some embodiments comprising a pressure chamber engaging athermocycler block;

FIG. 38 is a cross-sectional view illustrating a pressure clamp systemaccording to some embodiments comprising a vacuum assist system;

FIG. 39 is a cross-sectional view illustrating a pressure clamp systemaccording to some embodiments comprising a pressure chamber engaging athermocycler block and a microplate;

FIG. 40 is a cross-sectional view illustrating a pressure clamp systemaccording to some embodiments comprising a pressure chamber and a reliefport;

FIG. 41 is an exploded cross-sectional view illustrating a pressureclamp system according to some embodiments comprising a heatabletransparent window;

FIG. 42 is a top perspective view illustrating an upright configuration,according to some embodiments, of a thermocycler system, an excitationsystem, a detection system, and a microplate;

FIG. 43 is a side view illustrating the upright configuration of thethermocycler system, the excitation system, the detection system, andthe microplate of FIG. 42;

FIG. 44 is a perspective view illustrating an inverted configuration,according to some embodiments, of a thermocycler system, an excitationsystem, a detection system, and a microplate;

FIG. 45 is an enlarged perspective view illustrating an excitationsystem according to some embodiments comprising a plurality of LEDexcitation sources;

FIG. 46 is an enlarged perspective view illustrating an excitationsystem according to some embodiments comprising a plurality of LEDexcitation sources;

FIG. 47 is a side view illustrating the inverted configuration of thethermocycler system, the excitation system, the detection system, andthe microplate of FIG. 44;

FIG. 48 is a perspective view illustrating an inverted configuration,according to some embodiments, of a thermocycler system, an excitationsystem comprising individually mirrored excitation sources, a detectionsystem, and a microplate;

FIG. 49 is an enlarged perspective view illustrating the excitationsystem comprising individually mirrored excitation sources of FIG. 48;

FIG. 50 is a graph exemplifying vignetting and shadowing relative toexcitation source position;

FIG. 51 is a graph exemplifying vignetting and shadowing and anillumination profile according to some embodiments;

FIG. 52 is a schematic view illustrating an excitation source comprisinga lens according to some embodiments;

FIG. 53 is a schematic view illustrating an excitation source comprisinga concave mirror according to some embodiments;

FIG. 54 is a schematic view illustrating an excitation source comprisinga concave mirror and a lens according to some embodiments;

FIG. 55 is a schematic view illustrating multiple excitation sourcesfocused to a point on a microplate according to some embodiments;

FIG. 56 is a schematic view illustrating multiple excitation sourcesfocused to multiple points to achieve a desired irradiance profileaccording to some embodiments;

FIG. 57 is a flow chart illustrating a manufacturing procedure ofpreloaded microplates according to some embodiments;

FIG. 58 is a flow chart illustrating the use of a database systemaccording to some embodiments;

FIG. 59 is a top perspective view illustrating a multipiece microplatein accordance with some embodiments;

FIG. 60 is an exploded perspective view illustrating the multipiecemicroplate of FIG. 59 in accordance with some embodiments;

FIG. 61 is a top view illustrating the multipiece microplate inaccordance with some embodiments;

FIG. 62 is a cross-sectional view of the multipiece microplate of FIG.61 taken along Line 62-62;

FIG. 63 is an enlarged cross-sectional view of cap portion and main bodyportion of the multipiece microplate of FIG. 62;

FIG. 64 is a top schematic view illustrating a loading distributionsystem comprising a conveyer, a plurality of dispensing stations, aplurality of robots, and a plurality of microplate hotels according tosome embodiments;

FIG. 65 is a perspective view illustrating a loading distribution systemaccording to some embodiments;

FIG. 66 is a side view illustrating a loading distribution systemaccording to some embodiments, comprising a dispensing device, a sourceplate and wash station, and a carriage;

FIG. 67 is a side view illustrating a loading distribution systemaccording to some embodiments, comprising a dispensing device, a sourceplate station, a wash station, and a carriage;

FIGS. 68( a)-(c) are top-plan views illustrating various uses of asource plate and wash pallet;

FIG. 69 is a top-plan view illustrating a ceiling mounted plate-handlingdevice adapted to retrieve a microplate from a hotel according to someembodiments;

FIG. 70 is a perspective view illustrating a carriage capable of holdinga microplate according to some embodiments;

FIG. 71 is a perspective view illustrating a table coupled to a carriageutilizing a spring allowing the table to float in X and Y axis withrespect to the carriage according to some embodiments;

FIG. 72 is a perspective view illustrating an embodiment of a locatingratchet adapted to hold a microplate on the table according to someembodiments;

FIG. 73 is a perspective view illustrating a lifting device to allow thetable to float in Z axis with respect to the carriage according to someembodiments;

FIG. 74 is a perspective view illustrating a pressure source adapted tocommunicate with a vacuum connection shoe according to some embodiments;

FIG. 75 is a perspective view illustrating of a loading distributionsystem comprising a pair of rails and a guide channel to lift the tableoff of the carriage according to some embodiments

FIG. 76 is a perspective view illustrating an air slide connecting thepair of rails and a guide channel according to some embodiments;

FIG. 77 is a perspective view illustrating a loading distribution systemcomprising the carriage, the table, and an alignment stage according tosome embodiments;

FIG. 78 is a perspective view illustrating a lifting stage adapted tolift a carriage according to some embodiments;

FIGS. 79( a)-(b) are perspective views illustrating a visual inspectionstation including a carriage alignment device according to someembodiments;

FIG. 80 is a top-plan view illustrating a table comprising a vacuumtrench and a gasket according to some embodiments;

FIG. 81 is a perspective view illustrating a dispensing device includinga plurality of dispensers according to some embodiments;

FIG. 82 is a perspective view illustrating a plate gripper robotaccording to some embodiments;

FIG. 83 is a perspective view illustrating a plate gripper robot,gripping a microplate in a lower jaw according to some embodiments;

FIGS. 84-90 are progressive perspective views illustrating a plategripper robot depositing and picking-up microplates from a table and/ora plate storage unit according to some embodiments;

FIG. 91 is a perspective view illustrating a source plate and washpallet according to some embodiments;

FIG. 92 is a perspective view illustrating a source plate and washstation, wherein a source plate and a washing tray each comprise arespective lid thereupon according to some embodiments;

FIG. 93 is a perspective view illustrating a source plate and washstation, wherein a de-lidded source plate allowing a dispensing deviceto access fluids stored in or on the source plate according to someembodiments;

FIG. 94 is a perspective view illustrating a source plate and washstation, wherein the source plate stays lidded and the washing tray canbe accessed by a dispensing device according to some embodiments;

FIG. 95 is a perspective view illustrating a source plate and washstation positioned to enable a robot gripper to access a lidded sourceplate according to some embodiments;

FIG. 96 is a perspective view illustrating a source plate and washstation positioned to a allow a dispensing station to access a sourceplate according to some embodiments;

FIG. 97 is a perspective view illustrating a source plate and washstation positioned to a allow a dispensing station to access the washingtray according to some embodiments;

FIG. 98 is a front-plan view illustrating a source plate and washstation in a wait position alongside a dispensing device and a conveyeraccording to some embodiments;

FIG. 99 is a front-plan view illustrating a source plate and washstation in a deployed position alongside a dispensing device and aconveyer according to some embodiments;

FIG. 100 is a perspective view illustrating a hotel and a movable entryguide according to some embodiments;

FIG. 101 is a process flow diagram illustrating a software command andcontrol architecture for a loading distribution system, according tosome embodiments;

FIG. 102 is an illustration a sample distribution mapping for an eightdispenser sample filler, according to some embodiments;

FIG. 103 is an illustration of using a dead row to preventcross-contamination in sample loadings from a filler according to someembodiments;

FIG. 104 is a top-plan view illustrating a robot accessing microplatehotels, source plate hotels, and a plurality of dispensing devicesaccording to some embodiments;

FIG. 105 is a top-plan view illustrating a mapping of fluid locations ofa 384-well source plate into a dispensing device comprising 96dispensers and further into a 6,144-well microplate according to someembodiments;

FIG. 106 is an exploded top perspective view illustrating a fillingapparatus comprising an intermediate layer according to someembodiments;

FIG. 107 is an exploded bottom perspective view illustrating the fillingapparatus comprising the intermediate layer according to someembodiments;

FIG. 108 is a cross-sectional view illustrating the filling apparatuscomprising the intermediate layer according to some embodiments;

FIG. 109 is a cross-sectional view illustrating the filling apparatuscomprising the intermediate layer and nodules according to someembodiments;

FIG. 110 is a top schematic view of the filling apparatus comprising theintermediate layer and nodules according to some embodiments;

FIG. 111 is a cross-sectional view illustrating the filling apparatuscomprising the intermediate layer, nodules, and sealing featureaccording to some embodiments;

FIG. 112 is a bottom perspective view of the intermediate layer of thefilling apparatus according to some embodiments;

FIG. 113 is an exploded top perspective view illustrating a clamp systemfor a filling apparatus according to some embodiments;

FIG. 114 is an exploded top perspective view illustrating a fillingapparatus comprising a vent layer according to some embodiments;

FIG. 115 is an exploded bottom perspective view illustrating the fillingapparatus comprising the vent layer according to some embodiments;

FIG. 116 is a cross-sectional view illustrating the filling apparatuscomprising the vent layer and a vent manifold according to someembodiments;

FIG. 117 is a top schematic view of the filling apparatus comprising thevent layer and vent apertures positioned between staging capillariesaccording to some embodiments;

FIG. 118 is a top schematic view of the filling apparatus comprising thevent layer and oblong vent apertures according to some embodiments;

FIG. 119 is a cross-sectional view illustrating the filling apparatuscomprising the vent layer and pressure bores according to someembodiments;

FIG. 120 is a perspective view illustrating a filling apparatuscomprising one or more assay input ports positioned on an end of aninput layer according to some embodiments;

FIG. 121 is a perspective view illustrating a filling apparatuscomprising one or more assay input ports positioned on a side of aninput layer according to some embodiments;

FIG. 122 is a perspective view illustrating a filling apparatuscomprising one or more assay input ports positioned on opposing sides ofan input layer according to some embodiments;

FIG. 123 is a perspective view with portions illustrated incross-section illustrating an assay input port according to someembodiments;

FIG. 124 is a cross-sectional view illustrating the filling apparatus ofFIGS. 120-123 according to some embodiments;

FIGS. 125-131 and 133 are cross-sectional views illustrating theprogressive filling of a microplate according to some embodiments;

FIG. 132 is a top schematic view of the filling apparatus comprisingreduced material areas for, at least in part, use in staking accordingto some embodiments;

FIGS. 134-139 are cross-sectional views illustrating the progressivefilling of a microplate using a filling apparatus employing fluidoverfill reservoirs according to some embodiments;

FIG. 140 is a cross-sectional view illustrating a filling apparatusemploying fluid overfill reservoirs disposed in an output layeraccording to some embodiments;

FIGS. 141( a)-(g) are top schematic views illustrating various possiblepositions of the staging capillaries relative to correspondingmicrofluidic channels according to some embodiments;

FIGS. 142( a)-(g) are cross-sectional views illustrating variouspossible positions and configurations microfluidic channels and stagingcapillaries according to some embodiments;

FIG. 143 is an exploded perspective view illustrating a fillingapparatus comprising a floating insert and cover according to someembodiments;

FIG. 144 is a cross-sectional view illustrating the filling apparatuscomprising the floating insert according to some embodiments;

FIG. 145 is an exploded perspective view illustrating a fillingapparatus comprising a floating insert according to some embodiments;

FIG. 146 is a cross-sectional view illustrating a floating insertaccording to some embodiments;

FIG. 147 is a cross-sectional view illustrating a floating insertcomprising post members according to some embodiments;

FIG. 148 is a cross-sectional view illustrating a floating insertcomprising tapered members according to some embodiments;

FIG. 149 is a cross-sectional view illustrating a floating insertcomprising tapered members and a flanged base portion according to someembodiments;

FIG. 150 is a cross-sectional view illustrating the floating insertcomprising tapered members and the flanged base portion inserted into acorresponding depression according to some embodiments;

FIG. 151 is a cross-sectional view illustrating the floating insertcomprising tapered members and the flanged base portion inserted intothe corresponding depression and assay flow therebetween according tosome embodiments;

FIG. 152 is a cross-sectional view illustrating the floating insertcomprising tapered members and the flanged base portion being forceddown onto the corresponding depression according to some embodiments;

FIGS. 153-155 are cross-sectional views illustrating the progressivefilling and release of assay from the filling apparatus illustrated inFIG. 145 according to some embodiments;

FIGS. 156 and 157 are cross-sectional views illustrating the filling andrelease of assay from a filling apparatus comprising weight membersaccording to some embodiments;

FIG. 158 is a perspective view illustrating a filling apparatuscomprising a surface wire assembly and reservoir pockets according tosome embodiments;

FIG. 159 is a cross-sectional view illustrating the filling apparatuscomprising the surface wire assembly according to some embodiments;

FIGS. 160-162 are cross-sectional views illustrating the progressivefilling of a plurality of staging capillaries according to someembodiments;

FIG. 163 is a perspective view illustrating a filling apparatuscomprising a surface wire assembly, a reservoir trough, and absorbentmember according to some embodiments;

FIG. 164 is a perspective view illustrating the filling apparatuscomprising the surface wire assembly, the reservoir trough, andabsorbent member further comprising a sloping portion according to someembodiments;

FIG. 165 is a perspective view illustrating a filling apparatuscomprising a surface wire assembly, reservoir pockets, and absorbentmembers according to some embodiments;

FIG. 166 is a perspective view illustrating the filling apparatuscomprising the surface wire assembly, reservoir pockets, and absorbentmembers further comprising a sloping overflow channel portion accordingto some embodiments;

FIG. 167 is a perspective view illustrating a funnel member comprisingan assay chamber according to some embodiments;

FIG. 168 is a perspective view illustrating a funnel member comprisingmultiple discrete assay chambers according to some embodiments;

FIG. 169 is a perspective view illustrating a funnel member comprisingmultiple discrete assay chambers according to some embodiments;

FIG. 170 is a cross-sectional view illustrating a funnel membercomprising a tip portion according to some embodiments;

FIG. 171 is a cross-sectional view illustrating a funnel membercomprising a tip portion and a wiper member according to someembodiments;

FIG. 172 is a cross-sectional view illustrating a funnel membercomprising a tip portion and a planar cavity according to someembodiments;

FIG. 173 is a cross-sectional view illustrating a funnel membercomprising a tip portion and a wiper member spaced apart from the tipportion according to some embodiments;

FIG. 174 is a bottom perspective view illustrating a funnel membercomprising multiple offset discrete assay chambers according to someembodiments;

FIG. 175 is a top plan view illustrating a funnel member comprisingmultiple offset discrete assay chambers and one or more aperturesaccording to some embodiments;

FIG. 176 is a cross-sectional view illustrating a funnel membercomprising multiple offset discrete assay chambers and one or moreapertures according to some embodiments;

FIG. 177 is a top perspective view illustrating a multipiece funnelmember comprising multiple offset discrete assay chambers and aninternal siphon passage according to some embodiments;

FIG. 178 is a cross-sectional view illustrating the multipiece funnelmember comprising multiple offset discrete assay chambers and theinternal siphon passage according to some embodiments;

FIG. 179 is an exploded top perspective view illustrating a multipiecefunnel member comprising portions separated generally verticallyaccording to some embodiments;

FIG. 180 is an exploded top perspective view illustrating a multipiecefunnel member comprising portions separated generally horizontallyaccording to some embodiments;

FIG. 181 is a cross-sectional view illustrating a sealing coveraccording to some embodiments;

FIG. 182 is a perspective view illustrating a sealing cover rollaccording to some embodiments;

FIG. 183 is a perspective view illustrating a manual sealing coverapplicator according to some embodiments;

FIG. 184 is a perspective view illustrating a fixture for use with amanual sealing cover applicator according to some embodiments;

FIG. 185 is a perspective view, with portions illustrated incross-section, illustrating the manual sealing cover applicatoraccording to some embodiments;

FIG. 186 is a side view, with portions illustrated in cross-section,illustrating the manual sealing cover applicator in a closed positionaccording to some embodiments;

FIG. 187 is a side view, with portions illustrated in cross-section,illustrating the manual sealing cover applicator in an opened positionaccording to some embodiments;

FIG. 188 is a perspective view illustrating an automated sealing coverapplicator employing a sealing cover roll according to some embodiments;

FIG. 189 is a perspective view, with portions removed for clarity,illustrating the automated sealing cover applicator employing thesealing cover roll according to some embodiments;

FIG. 190 is a cross-sectional view illustrating the automated sealingcover applicator employing the sealing cover roll according to someembodiments;

FIG. 191 is a perspective view illustrating a sealing cover rollcartridge according to some embodiments;

FIG. 192 is a cross-sectional view illustrating the sealing cover rollcartridge according to some embodiments;

FIG. 193 is a perspective view, with portions removed for clarity,illustrating the automated sealing cover applicator employing a singlesheet cartridge according to some embodiments;

FIG. 194 is a perspective view, with portions removed for clarity,illustrating a single sheet applicator assembly according to someembodiments;

FIG. 195 is a perspective view, with portions removed for clarity,illustrating a single cover cartridge according to some embodiments;

FIG. 196 is an enlarged cross-sectional view illustrating the singlecover cartridge according to some embodiments;

FIG. 197 is an exploded perspective view illustrating the single covercartridge according to some embodiments;

FIGS. 198-201 are cross-sectional views illustrating progressive stepsof applying a single sealing cover to a microplate according to someembodiments;

FIG. 202 is an exploded view illustrating an inverted configuration of apressure chamber according to some embodiments;

FIG. 203 is a cross-sectional view illustrating section A-A of thepressure chamber of FIG. 202 in combination with a thermocycler systemaccording to some embodiments;

FIG. 204 is a side view illustrating a clamp mechanism in a lockedcondition according to some embodiments;

FIG. 205 is a side view illustrating a clamp mechanism in an unlockedcondition according to some embodiments;

FIG. 206 is a bottom perspective view illustrating a clamp mechanism ina locked condition according to some embodiments;

FIG. 207 is a pneumatic diagram illustrating a pneumatic system for apressure chamber and a clamp mechanism according to some embodiments;

FIG. 208 is a perspective view illustrating the pneumatic system of FIG.207 according to some embodiments;

FIG. 209 is a flow diagram illustrating a method of clamping a chamberto a thermocycler system according to some embodiments;

FIG. 210 is a flow diagram illustrating a method of performing a leaktest on a chamber according to some embodiments;

FIG. 211 is a flow diagram illustrating a method of unclamping a chamberfrom a thermocycler system according to some embodiments;

FIG. 212 is a cross-sectional view illustrating an adjustable lens andcamera mount according to some embodiments; and

FIG. 213 is a flowchart illustrating a process for determining bias.

DESCRIPTION OF SOME EMBODIMENTS

The following description of some embodiments is merely exemplary innature and is in no way intended to limit the present teachings,applications, or uses. Although the present teachings will be discussedin some embodiments as relating to polynucleotide amplification, such asPCR, such discussion should not be regarded as limiting the presentteaching to only such applications.

The section headings and sub-headings used herein are for generalorganizational purposes only and are not to be construed as limiting thesubject matter described in any way.

High-Density Sequence Detection System

In some embodiments, a high density sequence detection system comprisesone or more components useful in an analytical method or chemicalreaction, such as the analysis of biological and other materialscontaining polynucleotides. Such systems are, in some embodiments,useful in the analysis of assays, as further described below. Highdensity sequence detection systems, in some embodiments, comprise anexcitation system and a detection system which can be useful foranalytical methods involving the generation and/or detection ofelectromagnetic radiation (e.g., visible, ultraviolet or infrared light)generated during analytical procedures. In some embodiments, suchprocedures include those comprising the use of fluorescent or othermaterials that absorb and/or emit light or other radiation underconditions that allow quantitative and/or qualitative analysis of amaterial (e.g., assays among those described herein). In someembodiments useful for polynucleotide amplification and/or detection, ahigh density sequence detection system can further comprise athermocycler. In some embodiments, a high density sequence system canfurther comprise microplate and components for, e.g., filling andhandling the microplate, such as a pressure clamp system. It will beunderstood that, although high density sequence detection systems aredescribed herein with respect to specific microplates, assays and otherembodiments, such systems and components thereof are useful with avariety of analytical platforms, equipment, and procedures.

Referring to FIG. 1, a high-density sequence detection system 10 isillustrated in accordance with some embodiments of the presentteachings. In some embodiments, high-density sequence detection system10 comprises a microplate 20 containing an assay 1000 (see FIGS. 26 and27), a thermocycler system 100, a pressure clamp system 110, anexcitation system 200, and a detection system 300 disposed in a housing1008.

In some embodiments, assay 1000 can comprise any material that is usefulin, the subject of, a precursor to, or a product of, an analyticalmethod or chemical reaction. In some embodiments for amplificationand/or detection of polynucleotides, assay 1000 comprises one or morereagents (such as PCR master mix, as described further herein); ananalyte (such as a biological sample comprising DNA, a DNA fragment,cDNA, RNA, or any other nucleic acid sequence), one or more primers, oneor more primer sets, one or more detection probes; components thereof;and combinations thereof. In some embodiments, assay 1000 comprises ahomogenous solution of a DNA sample, at least one primer set, at leastone detection probe, a polymerase, and a buffer, as used in a homogenousassay (described further herein). In some embodiments, assay 1000 cancomprise an aqueous solution of at least one analyte, at least oneprimer set, at least one detection probe, and a polymerase. In someembodiments, assay 1000 can be an aqueous homogenous solution. In someembodiments, assay 1000 can comprise at least one of a plurality ofdifferent detection probes and/or primer sets to perform multiplex PCR,which can be useful, for example, when analyzing a whole genome (e.g.,20,000 to 30,000 genes, or more) or other large numbers of genes or setsof genes.

Microplate

In some embodiments, a microplate comprises a substrate useful in theperformance of an analytical method or chemical reaction. In someembodiments, a microplate can comprise one or more material retentionregions, configured to hold or support a material (e.g., an assay, asdiscussed below, or other solid or liquid) at one or more locations onor in the microplate. In some embodiments, such material retentionregions can be wells, through-holes, hydrophilic spots or pads, and thelike. In some embodiments, such as shown in FIG. 2-19, materialretention regions comprise wells, as at 26. In some embodiments, suchwells can comprise a feature on or in the surface of the microplatewherein assay 1000 is contained at least in part by physical separationfrom adjacent features. Such well features can include, in someembodiments, depressions, indentations, ridges, and combinationsthereof, in regular or irregular shapes. In some embodiments amicroplate is single-use, wherein it is filled or otherwise used with asingle assay for a single experiment or set of experiments, and isthereafter discarded. In some embodiments, a microplate is multiple-use,wherein it can be operable for use in a plurality of experiments or setsof experiments.

Referring now to FIGS. 2-19, in some embodiments, microplate 20comprises a substantially planar construction having a first surface 22and an opposing second surface 24 (see FIG. 12-19). First surface 22comprises a plurality of wells 26 disposed therein or thereon. Theoverall positioning of the plurality of wells 26 can be referred to as awell array. Each of the plurality of wells 26 is sized to receive assay1000 (FIGS. 26 and 27). As illustrated in FIGS. 26 and 27, assay 1000 isdisposed in at least one of the plurality of wells 26 and sealing cover80 (FIG. 26) is disposed thereon (as will be discussed herein). In someembodiments, one or more of the plurality of wells 26 may not becompletely filled with assay 1000, thereby defining a headspace 1006(FIG. 26), which can define an air gap or other gas gap.

In some embodiments, the material retention regions of microplate 20 cancomprise a plurality of reaction spots on the surface of the microplate.In such embodiments, a reaction spot can be an area on the substratewhich localizes, at least in part by non-physical means, assay 1000. Insuch embodiments, assay 1000 can be localized in sufficient quantity,and isolation from adjacent areas on the microplate, so as to facilitatean analytical or chemical reaction (e.g., amplification of one or moretarget DNA) in the material retention region. Such localization can beaccomplished by physical and chemical modalities, including, forexample, physical containment of reagents in one dimension and chemicalcontainment in one or more other dimensions.

In some embodiments, the surface of the microplate 20 comprises anenhanced surface which can comprise a physical or chemical modality onor in the surface of the microplate so as to enhance support of, orfilling of, assay 1000 in a material retention region (e.g., a well or areaction spot). Such modifications can include chemical treatment of thesurface, or coating the surface. In some embodiments, such chemicaltreatment can comprise chemical treatment or modification of the surfaceof the microplate so as to form relatively hydrophilic and hydrophobicareas. In some embodiments, a surface tension array can be formedcomprising a pattern of hydrophilic sites forming reaction spots on ahydrophobic matrix, such that the hydrophilic sites can be spatiallysegregated by hydrophobic regions. Reagents delivered to the array canbe constrained by surface tension difference between hydrophilic andhydrophobic sites.

In some embodiments, the chemical modality can comprise chemicaltreatment or modification of the surface or other material of microplate20 so as to affix one or more components of assay 1000 to themicroplate. In such embodiments, assay 1000 can be affixed to microplate20, directly or indirectly, so that assay 1000 is operable for analysisor reaction, but is not removed or otherwise displaced from themicroplate prior to the analysis or reaction during routine handling ofthe microplate. In some embodiments, assay 1000 can be affixed to thesurface so as form a patterned array (immobilized reagent array) ofreaction spots. In some embodiments, an immobilization reagent array cancomprise a hydrogel affixed to the microplate. Such hydrogels caninclude, for example, cellulose gels, such as agarose and derivatizedagarose (e.g., low melting agarose, monoclonal anti-biotin agarose, andstreptavidin derivatized agarose); xanthan gels; synthetic hydrophilicpolymers, such as crosslinked polyethylene glycol, polydimethylacrylamide, polyacrylamide, polyacrylic acid (e.g., cross-linked withdysfunctional monomers or radiation cross-linking), and micellarnetworks; and combinations thereof.

In some embodiments, one or more components of assay 1000 can be affixedto microplate 20 by covalent or non-covalent bonding to the surface ofthe microplate. In certain embodiments, assay 1000 an be bonded,anchored or tethered to a second moiety (immobilization moiety) which,in turn, can be anchored to the surface of the microplate. In someembodiments, such anchoring is through a chemically releasable orcleavable moeity, such that assay 1000 can be released or made availablefor analysis or reaction after reacting with a cleaving reagent priorto, during, or after the microplate assembly. Such release methods caninclude a variety of enzymatic, or non-enzymatic means, such aschemical, thermal, or photolytic treatment. In some embodiments,chemical moieties for immobilization moieties can include thosecomprising carbamate, ester, amide, thiolester, (N)-functionalizedthiourea, functionalized maleimide, amino, disulfide, amide, hydrazone,streptavidin, avidin/biotin, and gold-sulfide groups.

Microplate Footprint

With reference to FIGS. 2-19, microplate 20 generally comprises a mainbody or substrate 28. In some embodiments, main body 28 is substantiallyplanar. In some embodiments, microplate 20 comprises an optional skirtor flange portion 30 disposed about a periphery of main body 28 (seeFIG. 2). Skirt portion 30 can form a lip around main body 28 and canvary in height. Skirt portion 30 can facilitate alignment of microplate20 on thermocycler block 102. Additionally, skirt portion 30 can provideadditional rigidity to microplate 20 such that during handling, filling,testing, and the like, microplate 20 remains rigid, thereby ensuringassay 1000, or any other components, disposed in each of the pluralityof wells 26 does not contaminate adjacent wells. However, in someembodiments, microplate 20 can employ a skirtless design (see FIGS. 3-5)depending upon user preference.

In order to facilitate use with existing equipment, robotic implements,and instrumentation, the footprint dimensions of main body 28 and/orskirt portion 30 of microplate 20, in some embodiments, can conform tostandards specified by the Society of Biomolecular Screening (SBS) andthe American National Standards Institute (ANSI), published January 2004(ANSI/SBS 3-2004). In some embodiments, the footprint dimensions of mainbody 28 and/or skirt portion 30 of microplate 20 are about 127.76 mm(5.0299 inches) in length and about 85.48 mm (3.3654 inches) in width.In some embodiments, the outside corners of microplate 20 comprise acorner radius of about 3.18 mm (0.1252 inches). In some embodiments,microplate 20 comprises a thickness of about 0.5 mm to about 3.0 mm. Insome embodiments, microplate 20 comprises a thickness of about 1.25 mm.In some embodiments, microplate 20 comprises a thickness of about 2.25mm. One skilled in the art will recognize that microplate 20 and skirtportion 30 can be formed in dimensions other than those specifiedherein.

Plurality of Wells

In order to increase throughput of genotyping, gene expression, andother assays, in some embodiments, microplate 20 comprises an increasedquantity of the plurality of wells 26 beyond that employed in priorconventional microplates. In some embodiments, microplate 20 comprises6,144 wells. According to the present teachings, microplate 20 cancomprise, but is not limited to, any of the array configurations ofwells described in Table 1.

TABLE 1 Total Number of Wells Rows × Columns Approximate Well Area 96  8× 12 9 × 9 mm 384 16 × 24 4.5 × 4.5 mm 1536 32 × 48 2.25 × 2.25 mm 345648 × 72 1.5 × 1.5 mm 6144 64 × 96 1.125 × 1.125 mm 13824  96 × 144 0.75× .075 mm 24576 128 × 192 0.5625 × 0.5625 mm 55296 192 × 288 0.375 ×0.375 mm 768 24 × 32 3 × 3 mm 1024 32 × 32 2.25 × 3 mm 1600 40 × 40 1.8× 2.7 mm 1280 32 × 40 2.25 × 2.7 mm 1792 32 × 56 2.25 × 1.714 mm 2240 40× 56 1.8 × 1.714 mm 864 24 × 36 3 × 3 mm 4704 56 × 84 1.257 × 1.257 mm7776  72 × 108 1 × 1 mm 9600 80 × 120 0.9 × .09 mm 11616 88 × 132 0.818× 0.818 mm 16224 104 × 156 0.692 × 0.692 mm 18816 112 × 168 0.643 ×0.643 mm 21600 120 × 180 0.6 × 0.6 mm 27744 136 × 204 0.529 × 0.529 mm31104 144 × 216 0.5 × 0.5 mm 34656 152 × 228 0.474 × 0.474 mm 38400 160× 240 0.45 × 0.45 mm 42336 168 × 252 0.429 × 0.429 mm 46464 176 × 2640.409 × 0.409 mm 50784 184 × 256 0.391 × 0.391 mmWell Shape

According to some embodiments, as illustrated in FIGS. 4 and 5, each ofthe plurality of wells 26 can be substantially equivalent in size. Theplurality of wells 26 can have any cross-sectional shape. In someembodiments, as illustrated in FIGS. 4, 26, and 27, each of theplurality of wells 26 comprises a generally circular rim portion 32(FIG. 4) with a downwardly-extending, generally-continuous sidewall 34that terminate at a bottom wall 36 interconnected to sidewall 34 with aradius. A draft angle of sidewall 34 can be used in some embodiments. Insome embodiments, the draft angle provides benefits including increasedease of manufacturing and minimizing shadowing (as discussed herein).The particular draft angle is determined, at least in part, by themanufacturing method and the size of each of the plurality of wells 26.In some embodiments, circular rim portion 32 can be about 1.0 mm indiameter, the depth of each of the plurality of wells 26 can be about0.9 mm, the draft angle of sidewall 34 can be about 1° to 5° or greaterand each of the plurality of wells 26 can have a center-to-centerdistance of about 1.125 mm. In some embodiments, the volume of each ofthe plurality of wells 26 can be about 500 nanoliters.

According to some embodiments, as illustrated in FIG. 5, each of theplurality of wells 26 comprises a generally square-shaped rim portion 38with downwardly-extending sidewalls 40 that terminate at a bottom wall42. A draft angle of sidewalls 40 can be used. Again, the particulardraft angle is determined, at least in part, by the manufacturing methodand the size of each of the plurality of wells 26. In some embodimentsof wells 26 of FIG. 5, generally square-shaped rim portion 38 can have aside dimension of about 1.0 mm in length, a depth of about 0.9 mm, adraft angle of about 1° to 5° or greater, and a center-to-centerdistance of about 1.125 mm, generally indicated at A (see FIG. 27). Insome embodiments, the volume of each of the plurality of wells 26 ofFIG. 5 can be about 500 nanoliters. In some embodiments, the spacingbetween adjacent wells 26, as measured at the top of a wall dividing thewells, is less than about 0.5 m. In some embodiments, this spacingbetween adjacent wells 26 is about 0.25 mm.

In some embodiments, and in some configurations, the plurality of wells26 comprising a generally circular rim portion 32 can provide advantagesover the plurality of wells 26 comprising a generally square-shaped rimportion 38. In some embodiments, during heating, it has been found thatassay 1000 can migrate through capillary action upward along edges ofsidewalls 40. This can draw assay 1000 from the center of each of theplurality of wells 26, thereby causing variation in the depth of assay1000. Variations in the depth of assay 1000 can influence the emissionoutput of assay 1000 during analysis. Additionally, during manufactureof microplate 20, in some cases cylindrically shaped mold pins used toform the plurality of wells 26 comprising generally circular rim portion32 can permit unencumbered flow of molten polymer thereabout. Thisunencumbered flow of molten polymer results in less deleterious polymermolecule orientation. In some embodiments, generally circular rimportion 32 provides more surface area along microplate 20 for improvedsealing with sealing cover 80, as is discussed herein.

Pressure Relief Bores

Referring now to FIGS. 6-9, in some embodiments, each of the pluralityof wells 26 of microplate 20 can comprise a pressure relief bore 44. Insome embodiments, pressure relief bore 44 is sized such that it does notinitially fill with assay 1000 due to surface tension. However, whenassay 1000 is heated during thermocycling, assay 1000 expands, therebyincreasing an internal fluid pressure in each of the plurality of wells26. This increased internal fluid pressure is sufficient to permit assay1000 to flow into pressure relief bore 44 as illustrated in FIG. 7,thereby minimizing the pressure exerted on sealing cover 80. In someembodiments, each of the plurality of wells 26 can have one or aplurality of pressure relief bores 44.

In some embodiments, as illustrated in FIGS. 8 and 9, pressure reliefbore 44 can be offset within each of the plurality of wells 26 so thateach of the plurality of wells 26 can be filled with assay 1000 or othermaterial 1004 via a spotting device 700 (FIG. 8) or a micro-piezodispenser 702 (FIG. 9). In some embodiments, a top edge 46 of pressurerelief bore 44 can be generally square and have minimal or no radius.This arrangement can reduce the likelihood that assay 1000 or othermaterial 1004 will enter pressure relief bore 44 prior to thermocycling.

Through-Hole Wells

Turning now to FIGS. 10, 33, and 36, in some embodiments, each of theplurality of wells 26 of microplate 20 comprises a plurality ofapertures 48 being sealed at least on one end by sealing cover 80. Insome embodiments, each of the plurality of apertures 48 is sealed on anopposing end with a foil seal 50, which can have a clear or opaqueadhesive. In these embodiments, foil seal 50 can be placed againstthermocycler block 102 to aid in thermal conductivity and distribution.

In some embodiments, a layer of mineral oil can be placed at the top ofeach of the plurality of apertures 48 before, or as an alternative to,placement of sealing cover 80 on microplate 20. In several of suchembodiments, the mineral oil can fill a portion of each of the pluralityof apertures 48 and provide an optical interface and can controlevaporation of assay 1000.

Grooves

Referring to FIGS. 11-15, in some embodiments, microplate 20 cancomprise grooves 52 and grooves 54 disposed about a periphery of theplurality of wells 26. In some embodiments, grooves 52 can have depthand width dimensions generally similar to the depth and width dimensionsof the plurality of wells 26 (FIGS. 12 and 13). In some embodiments,grooves 54 can have depth and width dimensions less than the depth andwidth dimensions of the plurality of wells 26 (FIGS. 14 and 15). In someembodiments, as illustrated in FIG. 12, additional grooves 56 can bedisposed at opposing sides of microplate 20. In some embodiments,grooves 52, 54, and 56 can improve thermal uniformity among theplurality of wells 26 in microplate 20. In some embodiments, grooves 52,54, and 56 can improve the sealing interface formed by sealing cover 80and microplate 20. Grooves 52, 54, and 56 can also assist in simplifyingthe injection molding process of microplate 20. In some embodiments, aliquid solution similar to assay 1000 can be disposed in grooves 52, 54,and 56 to, in part, improve thermal uniformity during thermocycling.

Alignment Features

In some embodiments, as illustrated in FIGS. 2, 3, 11, and 14,microplate 20 comprises an alignment feature 58, such as a cornerchamfer, a pin, a slot, a cut corner, an indentation, a graphic, orother unique feature that is capable of interfacing with a correspondingfeature formed in a fixture, reagent dispensing equipment, and/orthermocycler. In some embodiments, alignment feature 58 comprises a nubor protrusion 60 as illustrated in FIG. 14. Additionally, in someembodiments, alignment features 58 are placed such that they do notinterfere with sealing cover 80 or at least one of the plurality ofwells 26. However, locating alignment features 58 near at least one ofthe plurality of wells 26 can provide improved alignment with dispensingequipment and/or thermocycler block 102.

Thermally Isolated Portion

In some embodiments, as illustrated in FIGS. 16-19, microplate 20comprises a thermally isolated portion 62. Thermally isolated portion 62can be disposed along at least one edge of main body 28. Thermallyisolated portion 62 can be generally free of wells 26 and can be sizedto receive a marking indicia 64 (discussed in detail herein) thereon.Thermally isolated portion 62 can further be sized to facilitate thehandling of microplate 20 by providing an area that can be easilygripped by a user or mechanical device without disrupting the pluralityof wells 26.

Still referring to FIGS. 16-19, in some embodiments, microplate 20comprises a first groove 66 formed along first surface 22 and a secondgroove 68 formed along an opposing second surface 24 of microplate 20.First groove 66 and second groove 68 can be aligned with respect to eachother to extend generally across microplate 20 from a first side 70 to asecond side 72. First groove 66 and second groove 68 can be furtheraligned upon first surface 22 and second surface 24 to define a reducedcross-section 74 between thermally isolated portion 62 and the pluralityof wells 26. This reduced cross-section 74 can provide a thermalisolation barrier to reduce any heat sink effect introduced by thermallyisolated portion 62, which might otherwise reduce the temperature cycleof some of the plurality of wells 26.

Marking Indicia

In some embodiments, as illustrated in FIGS. 2, 16 and 17, microplate 20comprises marking indicia 64, such as graphics, printing, lithograph,pictorial representations, symbols, bar codes, handwritings or any othertype of writing, drawings, etchings, indentations, embossments or raisedmarks, machine readable codes (i.e. bar codes, etc.), text, logos,colors, and the like. In some embodiments, marking indicia 64 ispermanent.

In some embodiments, marking indicia 64 can be printed upon microplate20 using any known printing system, such as inkjet printing, padprinting, hot stamping, and the like. In some embodiments, such as thoseusing a light-colored microplate 20, a dark ink can be used to createmarking indicia 64 or vice versa.

In some embodiments, microplate 20 can be made of polypropylene and havea surface treatment applied thereto to facilitate applying markingindicia 64. In some embodiments, such surface treatment comprises flametreatment, corona treatment, treating with a surface primer, or acidwashing. However, in some embodiments, a UV-curable ink can be used forprinting on polypropylene microplates.

Still further, in some embodiments, marking indicia 64 can be printedupon microplate 20 using a CO₂ laser marking system. Laser markingsystems evaporate material from a surface of microplate 20. Because CO₂laser etching can produce reduced color changes of marking indicia 64relative to the remaining portions of microplate 20, in someembodiments, a YAG laser system can be used to provide improved contrastand reduced material deformation.

In some embodiments, a laser activated pigment can be added to thematerial used to form microplate 20 to obtain improved contrast betweenmarking indicia 64 and main body 28. In some embodiments, anantimony-doped tin oxide pigment can be used, which is easily dispersedin polymers and has marking speeds as high as 190 inches per second.Antimony-doped tin oxide pigments can absorb laser light and can convertlaser energy to thermal energy in embodiments where indicia are createdusing a YAG laser.

In some embodiments, marking indicia 64 can identify microplates 20 tofacilitate identification during processing. Furthermore, in someembodiments, marking indicia 64 can facilitate data collection so thatmicroplates 20 can be positively identified to properly correlateacquired data with the corresponding assay. Such marking indicia 64 canbe employed as part of Good Laboratory Practices (GLP) and GoodManufacturing Practices (GMP), and can further, in some circumstances,reduce labor associated with manually applying adhesive labels, manuallytracking microplates, and correlating data associated with a particularmicroplate.

In some embodiments, marking indicia 64 can assist in alignment byplacing a symbol or other machine-readable graphic on microplate 20. Anoptical sensor or optical eye 1491 (FIG. 204) can detect marking indicia64 and can determine a location of microplate 20. In some embodiments,such location of microplate 20 can then be adjusted to achieve apredetermined position using, for example, a drive system ofhigh-density sequence detection system 10, sealing cover applicator1100, or other corresponding systems.

In some embodiments, the type (physical properties, characteristics,etc.) of marking indicia employed on a microplate can be selected so asto reduce thermal and/or chemical interference during thermocyclingrelative to what might otherwise occur with other types of markingindicia (e.g., common prior indicia designs, such as adhesive labels).For example, adhesive labels can, in some circumstances, interfere(e.g., chemically interact) with one or more reagents (e.g., dyes) beingused.

Referring to FIG. 2, in some embodiments, a radio frequencyidentification (RFID) tag 76 can be used to electronically identifymicroplate 20. RFID tag 76 can be attached or molded within microplate20. An RFID reader (not illustrated) can be integrated into high-densitysequence detection system 10 to automatically read a uniqueidentification and/or data handling parameters of microplate 20.Further, RFID tag 76 does not require line-of-sight for readability. Itshould be appreciated that RFID tag 76 can be variously configured andused according to various techniques, such as those described incommonly-assigned U.S. patent application Ser. No. 11/086,069, entitled“SAMPLE CARRIER DEVICE INCORPORATING RADIO FREQUENCY IDENTIFICATION, ANDMETHOD” filed herewith.

Multi-Piece Construction

In some embodiments, such as illustrated in FIGS. 59-63, microplate 20can comprise a multi-piece construction. In some embodiments, microplate20 can comprise main body 28 and a separate cap portion 95 that can beconnected with main body 28. In some embodiments, cap portion 95 can besized and/or shaped to mate with main body 28 such that the combinationthereof results in a footprint that conforms to the above-described SBSand/or ANSI standards. Alternatively, main body 28 and/or cap portion 95can comprise non-standard dimensions, as desired.

Cap portion 95 can be coupled with main body 28 in a variety of ways. Insome embodiments, cap portion 95 comprises a cavity 96 (FIG. 63), suchas a mortis, sized and/or shaped to receive a support member 97, such asa tenon, extending from main body 28 to couple cap portion 95 with mainbody 28. In some embodiments, cavity 96 of cap portion 95 and supportmember 97 of main body 28 can comprise an interference fit or otherlocking feature, such as a hook member, to at least temporarily joinmain body 28 and cap portion 95 during assembly. In some embodiments,support member 97 of main body 28 can comprise a cap alignment feature98 that can interface with a corresponding feature 99 on cap portion 95to properly align cap portion 95 relative to main body 28. In someembodiments, cap portion 95 can comprise alignment feature 58 for use inlater alignment of microplate 20 as described herein. In someembodiments, alignment feature 58 can be disposed on main body 28 toreduce tolerance buildup caused by the interface of cap portion 95 andmain body 28.

In some embodiments, cap portion 95 can be formed directly on main body28, such as through over-molding. In such embodiments, main body 28 canbe placed within a mold cavity that generally closely conforms to mainbody 28 and defines a cap portion cavity generally surrounding supportmember 97 of main body 28. Over-molding material can then be introducedabout support member 97 within cap portion cavity to form cap portion 95thereon.

In some embodiments, cap portion 95 comprises marking indicia 64 on anysurface(s) thereon (e.g. top surface, bottom surface, side surface). Insome embodiments, cap portion 95 can comprise an enlarged print areathereon relative to embodiments employing first groove 66 (FIG. 16-19).In some embodiments, cap portion 95 can be made of a material differentfrom main body 28. In some embodiments, cap portion 95 can be made of amaterial that is particularly conducive to a desired form of printing ormarking, such as through laser marking. In some embodiments, alaser-activated pigment can be added to the material used to form capportion 95 to obtain improved contrast between marking indicia 64 andcap portion 95. In some embodiments, an antimony-doped tin oxide pigmentcan be used. In some embodiments, cap portion 95 can be color-coded toaid in identifying a particular microplate relative to others.

In some embodiments, cap portion 95 can serve to provide a thermalisolation barrier through the interface of cavity member 96 and supportmember 97 to reduce any heat sink effect of cap portion 95 relative tomain body 28 to maintain a generally consistent temperature cycle of theplurality of wells 26. Cap portion 95 can be made, for example, of anon-thermally conductive material, such as one or more of those setforth herein, to, at least in part, help to thermally isolate capportion 95 from main body 28.

In some embodiments, cap portion 95 can serve to conceal any injectionmolding gates coupled to support member 97 during molding. Duringmanufacturing, as such gates are removed from any product, aestheticvariations can result. Any such aesthetic variations in main body 28 canbe concealed in some embodiments using cap portion 95. In some case,injection-molding gates can lead to a localized increase inflourescence. In some embodiments, such localized increase inflourescence can be reduced using cap portion 95.

Microplate Material

In some embodiments, microplate 20 can comprise, at least in part, athermally conductive material. In some embodiments, a microplate, inaccordance with the present teachings, can be molded, at least in part,of a thermally conductive material to define a cross-plane thermalconductivity of at least about 0.30 W/mK or, in some embodiments, atleast about 0.58 W/mK. Such thermally conductive materials can provide avariety of benefits, such as, in some cases, improved heat distributionthroughout microplate 20, so as to afford reliable and consistentheating and/or cooling of assay 1000. In some embodiments, thisthermally conductive material comprises a plastic formulated forincreased thermal conductivity. Such thermally conductive materials cancomprise, for example and without limitation, at least one ofpolypropylene, polystyrene, polyethylene, polyethyleneterephthalate,styrene, acrylonitrile, cyclic polyolefin, syndiotactic polystyrene,polycarbonate, liquid crystal polymer, conductive fillers or plasticmaterials; and mixtures or combinations thereof. In some embodiments,such thermally conductive materials include those known to those skilledin the art with a melting point greater than about 130° C. For example,microplate 20 can be made of commercially available materials such asRTP199X104849, COOLPOLY E1201, or, in some embodiments, a mixture ofabout 80% RTP199X104849 and 20% polypropylene.

In some embodiments, microplate 20 can comprise at least one carbonfiller, such as carbon, graphite, impervious graphite, and mixtures orcombinations thereof. In some cases, graphite has an advantage of beingreadily and cheaply available in a variety of shapes and sizes. Oneskilled in the art will recognize that impervious graphite can benon-porous and solvent-resistant. Progressively refined grades ofgraphite or impervious graphite can provide, in some cases, a moreconsistent thermal conductivity.

In some embodiments, one or more thermally conductive ceramic fillerscan be used, at least in part, to form microplate 20. In someembodiments, the thermally conductive ceramic fillers can comprise boronnitrate, boron nitride, boron carbide, silicon nitride, aluminumnitride, and mixtures or combinations thereof.

In some embodiments, microplate 20 can comprise an inert thermallyconductive coating. In some embodiments, such coatings can includemetals or metal oxides, such as copper, nickel, steel, silver, platinum,gold, copper, iron, titanium, alumina, magnesium oxide, zinc oxide,titanium oxide, and mixtures thereof.

In some embodiments, microplate 20 comprises a mixture of a thermallyconductive material and other materials, such as non-thermallyconductive materials or insulators. In some embodiments, thenon-thermally conductive material comprises glass, ceramic, silicon,standard plastic, or a plastic compound, such as a resin or polymer, andmixtures thereof to define a cross-plane thermal conductivity of belowabout 0.30 W/mK. In some embodiments, the thermally conductive materialcan be mixed with liquid crystal polymers (LCP), such as wholly aromaticpolyesters, aromatic-aliphatic polyesters, wholly aromaticpoly(ester-amides), aromatic-aliphatic poly(ester-amides), aromaticpolyazomethines, aromatic polyester-carbonates, and mixtures thereof. Insome embodiments, the composition of microplate 20 can comprise fromabout 30% to about 60%, or from about 38% to about 48% by weight, of thethermally conductive material.

The thermally conductive material and/or non-thermally conductivematerial can be in the form of, for example, powder particles, granularpowder, whiskers, flakes, fibers, nanotubes, plates, rice, strands,hexagonal or spherical-like shapes, or any combination thereof. In someembodiments, the microplate comprises thermally conductive additiveshaving different shapes to contribute to an overall thermal conductivitythat is higher than any one of the individual additives alone.

In some embodiments, the thermally conductive material comprises apowder. In some embodiments, the particle size used herein can bebetween 0.10 micron and 300 microns. When mixed homogeneously with aresin in some embodiments, powders provide uniform (i.e. isotropic)thermal conductivity in all directions throughout the composition of themicroplate.

As discussed above, in some embodiments, the thermally conductivematerial can be in the form of flakes. In some such embodiments, theflakes can be irregularly shaped particles produced by, for example,rough grinding to a desired mesh size or the size of mesh through whichthe flakes can pass. In some embodiments, the flake size can be between1 micron and 200 microns. Homogenous compositions containing flakes can,in some cases, provide uniform thermal conductivity in all directions.

In some embodiments, the thermally conductive material can be in theform of fibers, also known as rods. Fibers can be described, among otherways, by their lengths and diameters. In some embodiments, the length ofthe fibers can be, for example, between 2 mm and 15 mm. The diameter ofthe fibers can be, for example, between 1 mm and 5 mm. Formulations thatinclude fibers in the composition can, in some cases, have the benefitof reinforcing the resin for improved material strength.

In some embodiments, microplate 20 can comprise a material comprisingadditives to promote other desirable properties. In some embodiments,these additives can comprise flame-retardants, antioxidants,plasticizers, dispersing aids, marking additives, and mold-releasingagents. In some embodiments, such additives are biologically and/orchemically inert.

In some embodiments, microplate 20 comprises, at least in part, anelectrically conductive material, which can improve reagent dispensingalignment. In this regard, electrically conductive material can reducestatic build-up on microplate 20 so that the reagent droplets will notgo astray during dispensing. In some embodiments, a voltage can beapplied to microplate 20 to pull the reagent droplets into apredetermined position, particularly with a co-molded part where thebottom section can be electrically conductive and the sides of theplurality of wells 26 may not be electrically conductive. In someembodiments, a voltage field applied to the electrically conductivematerial under the well or wells of interest can pull assay 1000 intothe appropriate wells.

In some embodiments, microplate 20 can be made, at least in part, ofnon-electrically conductive materials. In some embodiments,non-electrically conductive materials can at least in part comprise oneor more of crystalline silica (3.0 W/mK), aluminum oxide (42 W/mK),diamond (2000 W/mK), aluminum nitride (150-220 W/mK), crystalline boronnitride (1300 W/mK), and silicon carbide (85 W/mK).

Microplate Molding

In some embodiments, microplate 20 can be molded by first extruding amelt blend comprising a mixture of a polymer and one or more thermallyconductive materials and/or additives. In some embodiments, the polymerand thermally conductive additives can be fed into a twin-screw extruderusing a gravimetric feeder to create a well-dispersed melt blend. Insome embodiments, the extruded melt blend can be transferred through awater bath to cool the melt blend before being pelletized and dried. Thepelletized melt blend can then be heated above its melting point by aninjection molding machine and then injected into a mold cavity. The moldcavity can generally conform to a desired shape of microplate 20. Insome embodiments, the injection-molding machine can cool the injectedmelt blend to create microplate 20. Finally, microplate 20 can beremoved from the injection-molding machine.

In some embodiments, two or more material types of pellets can be mixedtogether and the combination then placed in the injection moldingmachine to be melt blended during the injection molding process. In someembodiments, microplate 20 can be molded by first receiving pelletmaterial from a resin supplier; drying the pellet material in a resindryer; transferring the dried pellet material with a vacuum system intoa hopper of a mold press; molding microplate 20; trimming any resultantgates or flash; and packaging microplate 20. In some embodiments, themold cavity can be centrally gated along the second surface 24 ofmicroplate 20. In some embodiments, the mold cavity can be gated along aperimeter of main body 28 and/or skirt portion 30 of microplate 20.

Microplate Spotting, Filling, and Sealing

In some embodiments, one or more devices can be used to facilitate theplacement of one or more components of assay 1000 within at least someof the plurality of wells 26 of microplate 20.

Microplate Spotting

In some embodiments, as illustrated in FIG. 57, microplate 20 can bepreloaded with at least some component materials of assay 1000, such asreagents. In some embodiments, as described further herein, suchreagents can comprise at least one primer and at least one detectionprobe. In some embodiments, such reagents can comprise elementsfacilitating analysis of a whole genome or a portion of a genome. Stillfurther, in some embodiments, such reagents can comprise buffers and/oradditives useful for coating, stability, enhanced rehydration,preservation, and/or enhanced dispensing of reagents.

In some embodiments, such reagents can be delivered (e.g. spotted) intoat least one of the plurality of wells 26 of microplate 20 in verysmall, e.g. nanoliter, increments using a spotting device 700 (FIG. 8).In some embodiments, spotting device 700 employs one or morepiezoelectric pumps, acoustic dispersion, liquid printers, micropiezodispensers, or the like to deliver such reagents to each of theplurality of wells. In some embodiments, spotting device 700 employs anapparatus and method like or similar to that described in commonlyassigned U.S. Pat. Nos. 6,296,702, 6,440,217, 6,579,367, and 6,849,127,issued to Vann et al.

According to some embodiments, in operation, as schematicallyillustrated in FIG. 57, reagents, e.g. in an aqueous form or bead form,can be stored on one or more storage plates 704 in a high-humiditystorage unit 706. In some embodiments, high-humidity storage unit 706can comprise a relative humidity in the range of about 70-100%. However,in some embodiments, high-humidity storage unit 706 can comprise arelative humidity in the range of about 70-85%. The bead form can belike or similar to that described in commonly assigned U.S. Pat. No.6,432,719 to Vann et al. Some of the plurality of storage plates 704 canbe moved out of high-humidity storage unit 706, as indicated by 708, andcan be placed onto spotting device 700, as indicated by 710. A separateunspotted microplate 712 can then be moved out of a low-humidity storageunit 714, as indicated by 716. In some embodiments, low-humidity storageunit 714 can comprise a relative humidity in the range of about 0-30%.Unspotted microplate 712 can then be placed on spotting device 700, asindicated by 718. Reagents from storage plate 704 can then be spottedonto at least some of the plurality of wells 26 on unspotted microplate712. Once at least some of the plurality of wells 26 are spotted, thespotted microplate 720 can then be moved from spotting device 700, asindicated by 722. Spotted microplate 720 can then be moved to anoptional quality-control station 724, as indicated by 726. Afterquality-control station 724, spotted microplate 720 can then be movedback to low-humidity storage unit 714, as indicated by 728. Thisprocedure of spotting microplates 20 can continue until a desired number(e.g. all) of microplates in storage unit 714 have been spotted withreagents from storage plate 704. It should be noted that unspottedmicroplate 712 and spotted microplate 720 are each similar to microplate20, however different numerals are used for simplicity in the abovedescription.

In some embodiments, the spots of reagents on spotted microplate 720 canbe partially or fully dried down, as desired, in the low-humidity ofstorage unit 714. In some embodiments, storage unit 714 can also beheated to facilitate this drying. Once the microplates from storage unit714 have been spotted with reagents from storage plate 704, storageplate 704 can be removed and designated as a used storage plate 730.Used storage plate 730 can be removed from spotting device 700 asindicated by 732. Used storage plate 730 can be returned tohigh-humidity storage unit 706 as indicated by 734. The process cancontinue as the next storage plate 704 is moved out of high-humiditystorage unit 706 and into spotting device 700. In some embodiments, thisnext storage plate 704 can contain a different set of reagents. Theaforementioned process can then be repeated, as desired. This processcan continue until all of the plurality of wells 26 on spottedmicroplate 720 have been spotted or, in some cases, a portion of theplurality of wells 26 have been spotted, while leaving the remainingwells 26 empty.

It should be appreciated that this preloading process can vary asdesired to accommodate user needs. For instance, in some embodiments,the reagents spotted in each of the plurality of wells 26 can beencapsulated with a material. Such encapsulation can prevent or reducemoisture at room temperature from interacting with the reagents. In someembodiments, each of the plurality of wells 26 can be spotted severaltimes with reagents, such as for multiplex PCR. In some embodiments,these multiple spotted reagents can form layers. In some embodiments ofthis preloading process, primer sets and detection probes for a wholegenome can be spotted from storage plates 704 onto spotted microplate720. In other embodiments, a portion of a genome, or subsets of selectedgenes, can be spotted from source plates 704 onto spotted microplate720.

In some embodiments, spotted microplate 720 can be sealed with aprotective cover, stored, and/or shipped to another location. In someembodiments, the protective cover is releasable from spotted microplate720 in one piece without leaving adhesive residue on spotted microplate720. In some embodiments, the protective cover is visibly different(e.g., a different color) from sealing cover 80 to aid in visualidentification and for ease of handling.

In some embodiments, the protective cover can be made of a materialchosen to reduce static charge generation upon release from spottedmicroplate 720. When it is time for spotted microplate 720 to be used,the package seal can be broken and the protective cover can be removedfrom spotted microplate 720. In some embodiments, the protective covercan be a pierceable film, a slitted film, or a duckbilled closure to, atleast in part, reduce contamination and/or evaporation. An analyte (sucha biological sample comprising DNA) can then be added to spottedmicroplate 720, along with other materials such as PCR master mix, toform assay 1000 in at least some of the plurality of wells 26. Spottedmicroplate 720 can then be sealed with sealing cover 80 as describedabove. High-density sequence detection system 10 can then be actuated tocollect and analyze data.

In some embodiments, the filling apparatus comprises a device fordepositing (e.g., spotting or spraying) of assay 1000 to specific wells,wherein one or more of the plurality of wells 26 of microplate 20contains a different assay material than other wells 26 of microplate20. In some embodiments, the device can include piezoelectric pumps,acoustic dispersion, liquid printers, or the like. According to someembodiments, a pin spotter can be employed, such as described in PCTPublication No. WO 2004/018104. In some embodiments, a fiber and/orfiber-array spotter can be employed, such as described in U.S. Pat. No.6,849,127.

In some embodiments, the filling apparatus comprises a device fordepositing assay 1000 to a plurality of wells, wherein two or more wellscontain the same assay material. In some embodiments, microplate 20comprises two more groups of wells 26. Each of the groups of wells 26can comprise a different assay material than at least one other group ofwells 26 on microplate 20.

Loading Distribution System

Referring to FIG. 64, a loading distribution system 800 comprising aconveyer or a track 802 can be used to set up an expandable and flexiblemicroplate loading distribution system. For example, FIG. 64 depictsfour dispensing devices 814, 816, 818, and 820, disposed adjacent acorresponding source plate and wash station 814 a, 816 a, 818 a, and 820a, respectively. Dispensing devices 814, 816, 818, and 820 can eachcomprise a plurality of dispensers, for example, 24-dispensers,48-dispensers, 96-dispensers, 384-dispensers. FIG. 81 is a perspectiveview illustrating dispensing device 814 including a plurality ofdispensers 868, for example, in a SBS standard micro-titer format. Oneor more of dispensing devices 814, 816, 818, and 820 can comprise, forexample, the Aurora Scout MPD (MultiTip Piezo Dispenser) available fromAurora Discovery as, for example, a 96-tip dispensing device and/or a384-tip dispensing device. In some embodiments, the dispensing devicecan comprise at least 96 dispensing tips in loading distribution system800. The dispensing device can comprise, for example, at least 96dispensing tips, at least 384 dispensing tips, at least 768 dispensingtips, at least 1536 dispensing tips, or more. The dispensing device cancomprise a plurality of dispensers and each dispenser can comprise apiezo-electric dispenser. The dispensing device in loading distributionsystem 800 can comprise a plurality of dispensers and a respectiveplurality of storage reservoirs. Each dispenser can be designed todispense a first volume of fluid per dispensing action, and eachreservoir can be adapted to store many times the first volume, forexample, at least 15 times the first volume, at least 25 times the firstvolume, at least 50 times the first volume, or at least 100 times thefirst volume.

In some embodiments, each of the plurality of dispensers can be adaptedto dispense about 100 nanoliters of liquid or fluid, per dispensingaction. The dispensing device can comprise a plurality of spottingdevices. The dispensing devices can comprise, for example,piezo-electric devices, acoustic devices, ink-jet devices, pump-actiondevices, pin spotters, or the like, or a combination thereof.

In some embodiments, the number of dispensing devices 814, 816, 818, and820 disposed around a conveyer 802 can be increased or decreased so asto address a desired throughput target. In some embodiments, conveyer802 can expand (be lengthened) in an X-direction. This can allow moredispensing devices to be disposed around conveyer 802. Conveyer 802 cancomprise a track, for example, SuperTrak™ available from ATS AutomationTooling Systems Inc. However, it should be understood that other trackscan be used.

In some embodiments, loading distribution system 800 can comprise a loadposition 806 on conveyer 802. Loading distribution system 800 cancomprise an unload position 808 on conveyer 802. Load position 806 andunload position 808 can, according to some embodiments, be a sameposition along conveyer 802.

The plurality of stations can also include, for example, one or more ofan inspection station, a plurality of inspection stations, a trackingstation, an identifying tag reader station, or the like, as furtherdescribed herein. According to some embodiments and as further describedbelow, the table described herein can comprise a plurality of tables,with the number of tables, and corresponding carriages if used, beinggreater than or equal to the number of processing stations. In someembodiments, the plurality of processing stations in loadingdistribution system 800 can comprise an inspection station adapted tocheck an alignment of a microplate on the table. The inspection stationcan comprise, for example, one or more of a camera, a CCD, a laser, apattern analyzer, an edge analyzer, and a combination thereof. Theplurality of processing stations can comprise, for example, aninspection station adapted to perform a quality control analysis of aspot disposed on the microplate, wherein the inspection station cancomprise, for example, one or more of a camera, a CCD, a laser, apattern analyzer, an edge analyzer, and a combination thereof. In someembodiments, loading distribution system 800 can further comprise, forexample, a tracking device adapted to track dispensation of fluid fromthe dispensing device. The tracking device can track a microplate and beadapted to determine whether and which locations of a microplate havebeen processed, spotted, or otherwise prepared. The tracking device can,in some embodiments, be adapted to track the use of components of anassay. The tracking device can be adapted, for example, to communicatewith an identifying tag reader or with an identifying tag to track theprogress of a preparation procedure, for example, to track loadingand/or spotting operations at each of many loading and/or spottingsites. The tracking device can be adapted to communicate with machineindicia reader 804 and inspection station 810 illustrated in FIG. 64. Insome embodiments, a dispensing device can comprise a plurality ofdispensing devices and the tracking device can be adapted to trackdispensation of fluids from each of the dispensing devices to amicroplate. Methods of tracking are further discussed in more detailbelow.

In some embodiments, the plurality of processing stations can comprise atracking station, for example, an identifying tag reader station adaptedto read marking indicia 64 disposed on or in microplate 20. Theidentifying tag can be a bar code, a two-dimensional barcode, or othermarking indicia reader station adapted to read the identifying tag. Thereader station can comprise a reader device or apparatus appropriate tothe type of marking indicia employed, e.g., a bar code reader. Theidentifying tag can, in some embodiments, be a radio frequencyidentification (RFID) tag and the reader station can comprise a RFIDreader. In some embodiments, a marking indicia reader station in loadingdistribution system 800 can comprise one or more of a bar code reader, aone-dimensional bar code reader, a two-dimensional bar code reader, andan RFID reader. In some embodiments, a marking indicia reader station inloading distribution system 800 can be adapted to read marking indiciaon the same surface of the microplate that can engage the table when themicroplate is on the table.

In some embodiments, loading distribution system 800 can comprise amachine indicia reader 804 disposed along conveyer 802. Machine indiciareader 804 can, according to some embodiments, comprise a plurality ofmachine indicia readers, one each disposed prior to every dispensingdevice along conveyer 802. In some embodiments, machine indicia reader804 can be disposed past load position 806 along conveyer 802.

In some embodiments, a method of tracking a microplate is provided. Themethod can comprise, for example, a first dispensing operation thatcomprises spotting components of an assay to one or more locations ormaterial retention regions of a microplate, for example, one or morewells of a multiwell microplate, to form a partially loaded microplate.Each well can be spotted with a different set of components of adifferent respective assay. The method can comprise storing informationabout the at least partially loaded microplate by writing informationinto a memory using a value of the machine-readable identifier as anindex. The method can comprise storing information about the at leastpartially loaded microplate by writing information into a memory that isaddressable by a value associated with the machine-readable identifier.The stored information can comprise information pertaining to the wellsand which wells have been spotted and with what respective components ofan assay. By tracking such information, subsequent dispensing operationscan be directed to wells that have not been spotted and assay componentsthat have not yet been spotted into respective wells.

In some embodiments, the method of tracking can comprise subjecting amicroplate to two or more, for example, five or more, dispensingoperations and to two or more, for example, five or more, informationreading steps with at least one information reading step being conductedprior to or subsequent to each dispensing operation. According to someembodiments, the method of tracking can comprise a reading step followedby a plurality of dispensing operations at a respective plurality ofdispensing stations. The method can comprise storing information aboutthe at least partially loaded microplate by writing information to theradio frequency identification tag. The method can comprise: readinginformation from a machine-readable identifier on a microplate;subjecting the microplate to a first dispensing operation by a firstmulti-tip dispenser to at least partially load one or more materialretention regions of the microplate and form an at least partiallyloaded microplate; storing information about the at least partiallyloaded microplate; reading the information stored about the at leastpartially loaded microplate; and determining, based on the informationread about the at least partially loaded microplate, whether to subjectthe microplate to a subsequent dispensing operation by second multi-tipdispenser that differs from the first multi-tip dispenser. Thedetermining can comprise determining that the at least partially loadedmicroplate should be subjected to a subsequent dispensing operation, andthe method can then further comprise subjecting the microplate to anadditional dispensing operation by the second multi-tip dispenser, tofurther load the microplate.

The method of tracking can be used in connection with a systemcomprising a first multi-tip dispenser located at a first station, asecond multi-tip dispenser located at a second station, and a conveyerdevice connecting the two stations. The method can comprise conveyingthe microplate from the first station to the second station, along, on,or with, the conveyer device. The conveyer device can comprise, forexample, a track and/or a belt or chain. The conveyer device illustratedin FIGS. 64 and 65 comprises a track along which a carriage and tablecan ride or traverse.

The method of tracking can comprise, for example, reading theinformation stored about the at least partially loaded microplate byreading the information at a third station. The third station can belocated between the first station and the second station, along theconveyer device, or it can be located upstream or downstream of both thefirst and second stations. The first station and the second station canbe located adjacent each other along a track and the method can comprisedisposing the microplate on a carriage and conveying the carriage alongthe track from the first station to the second station.

In some embodiments, and as described further below, a system controller982 (FIG. 101) can manage and track microplates at various locations.Locations for a microplate can comprise, for example, in one or moreplate storage units, in or on one or more tables, or in one or more jawsof one or more plate handling devices. In some embodiments, systemcontroller 982 (FIG. 101) can, for example, manage and track microplatesat various locations in loading distribution system 800 (FIGS. 64 and65). Locations for a microplate can comprise, for example, in one ormore plate storage units, in or on one or more tables, or in one or morejaws of one or more plate handling devices. In some embodiments, systemcontroller 982 (FIG. 101) can, for example, manage and track sourceplates at various locations in loading distribution system 800 (FIGS. 64and 65). Locations for a source plate can comprise, for example, in asource plate storage unit like an incubator, in one or more source plateholders, or in one or more grippers of one or more source plate handlingdevices. System controller 982 described below with reference to FIG.101 can also, for example, track and trace the contents of one or moredispensers, each disposed in one or more respective dispensing devices.For example, system controller 982 can track and trace the contents ofone or more dispensers, each disposed in one or more respectivedispensing devices.

With reference to the perspective views of FIGS. 64 and 65, a number ofthe above-described features of the present teachings can be seenembodied in a high-throughput system for fabricating a microplate.Generally, conveyer 802 transports, in serial fashion, empty microplatesfrom a hotel or storage unit 828 to a position adjacent a load position806. Handling device 830 places the microplate on a table and carriageassembly for movement along conveyer 802. The microplate is then movedby the table and carriage assembly along conveyer 802 to machine indiciareader 804. The method of tracking can comprise scanning indicia on thebottom of the microplate. This operation can serve, for example, toensure that the card has been properly placed on the table and to readidentifying information into a control computer (not illustrated). Next,the table translates the microplate to dispensing stations 820, 818,816, 814, serially, for spotting operations.

Having received components of an assay from the dispensing stations, themicroplate can then be advanced to a position below an inspectionstation 810 that inspects each well of the microplate for the presenceof spotted components of an assay. If the inspection operations indicatethat the microplate has been properly loaded with components of anassay, the microplate is then moved along conveyer 802 to an unloadposition 808 where the microplate can be unloaded, for example, byhandling device 830, and moved back to the storage unit 828. If afailure is indicated, on the other hand, unloading at unload position808 can comprise depositing the microplate in a reject bin.

In a subsequent operation, for example, after a new set of respectiveassay components has been aspirated or loaded in dispensing heads ofdispensing stations 820, 818, 816, and 814, a partially loadedmicroplate can again be moved by handling device 830 onto a table of acarriage on conveyer 802, and then conveyed again to machine indiciareader 804. The method of tracking can then comprise reading informationstored about the microplate as a result of previous quality controlinspection at inspection station 810 and indexed by marking indicia onthe microplate. If further spotting of assay components is required, themicroplate can then be conveyed to dispensing stations 820, 818, 816,814 for further dispensing operations, this time with the newly-loadedassay components. After the further dispensing operations, the procedurecan be repeated, starting, for example, with another quality controlinspection at inspection station 810. Stored information correspondingto a marking indicia can be compared to predetermined values todetermine whether additional spotting is needed or whether themicroplate has been completely spotted with all desired assaycomponents.

According to some embodiments, the method of tracking can use a controlcomputer (not illustrated) that can integrate the operation of thevarious assemblies, for example through a program written in an eventdriven language such as LABVIEW® or LABWINDOWS® (National InstrumentsCorp., Austin, Tex.). In particular, the LABVIEW software provides ahigh level graphical programming environment for controllinginstruments. U.S. Pat. Nos. 4,901,221; 4,914,568; 5,291,587; 5,301,301;5,301,336; and 5,481,741 (each expressly incorporated herein in itsentirety by reference) disclose various aspects of the LABVIEW graphicalprogramming and development system. The graphical programmingenvironment disclosed in these patents allows a user to define programsor routines by block diagrams, or “virtual instruments.” As this isdone, machine language instructions are automatically constructed whichcharacterize an execution procedure corresponding to the displayedprocedure. Interface cards for communicating the computer with the motorcontrollers are also available commercially, for example, from NationalInstruments Corp.

In some embodiments, loading distribution system 800 can comprise aninspection station 810 disposed along conveyer 802. Inspection station810 can comprise, according to some embodiments, a plurality ofinspection stations, one disposed after each dispensing device alongconveyer 802. In some embodiments, a single inspection station 810 canbe disposed after all the dispensing devices along conveyer 802.

In some embodiments, loading distribution system 800 can comprise aplate-handling device 830 disposed on a plate-handling device pathway832 to access a storage unit 828 adapted to store microplates. Storageunit 828 can also be called a hotel. Loading distribution system 800 cancomprise a source plate-handling device 822. Source plate-handlingdevice 822 can be disposed on a source plate-handling device pathway 824to access a source plate storage unit 826 housing a plurality of sourceplates (not illustrated). Source plate storage unit 826 can comprise anincubator, for example, Kendro Cytomat 6001 available from KendroLaboratory Products. Storage unit 828 can comprise a hotel, for example,one or more 120 Nest Landscape Carousels. Plate-handling device 830 andsource plate-handling device 822 can each comprise a Select CompliantArticulated Robot Arm (SCARA) robot, respectively, available, forexample, from IAI America, Inc. The SCARA robots can be movable in4-axis or 5-axis. However, it should be understood that other robotmechanisms can be used.

In some embodiments, loading distribution system 800 can comprise astorage unit 828. Storage unit 828 can comprise a hotel, a carousel, oranother rack adapted to hold a plurality of microplates. In someembodiments, storage unit 828 can be accessible by the plate-handlingdevice so that the plate-handling device can retrieve microplates, forexample, one at a time, or store microplates therein, for example, oneat a time. Loading distribution system 800 can further comprise aplurality of microplates arranged in the storage unit.

As illustrated in FIG. 65, in some embodiments, dispensing devices 814,816, 818, and 820 can be disposed along conveyer 802 using a respectivedispensing device mount 814 c, 816 c, 818 c, and 820 c. Each dispensingdevice 814, 816, 818, and 820 can be disposed, for example, adjacent arespective alignment station 814 b, 816 b, 818 b, and 820 b. Alignmentstations 814 b, 816 b, 818 b, and 820 b can be adapted to move a table(not illustrated) in a Y-direction.

In some embodiments, when an alignment station is not provided to move atable in the Y-direction, a dispensing device can be moved in theY-direction to align a microplate disposed on the table with thedispensing device.

As illustrated in FIG. 66, in some embodiments, dispensing device 814can comprise a plurality of dispensers 868. A carriage 874 can bedisposed on conveyer 802. Carriage 874 can be positioned underdispensers 868, when dispensing of a fluid in or on microplate 20 isdesired. Microplate 20 can be disposed on a table 872. Table 872 cancomprise a vacuum chuck; see FIG. 80, adapted to hold microplate 20.Table 872 can move to align microplate with dispensers 868. Conveyer 802can translate carriage 874 away from the dispensing position. Carriage874 can move along conveyer 802.

In some embodiments, table 872 can be adapted to move along the Y-axisand the alignment stage can be adapted to align the microplate with thedispensing device. Table 872 can be adapted to be rotatable about theY-axis direction. As described herein, table 872 can comprise a vacuumchuck adapted to apply a vacuum to a surface of a microplate when amicroplate is disposed on the table. Loading distribution system 800 cancomprise a vacuum source in fluid communication with the vacuum chuck. Avacuum retainment valve can be disposed in fluid communication with thevacuum chuck and can be adapted to maintain a vacuum between the vacuumchuck and the surface of a microplate when a microplate is disposed onthe table, for example, when the vacuum chuck is not in fluidcommunication with the vacuum source. Loading distribution system 800can comprise a vacuum detector adapted to verify the formation of avacuum between the surface of a microplate disposed on the table, andthe vacuum chuck.

In some embodiments, loading distribution system 800 can furthercomprise an accessory carriage configured to engage a source platecomprising a source of fluids to be loaded into the spotting or otherdispensing station. The accessory carriage can be adapted to move thesource plate to the dispensing station for aspiration of the fluids fromthe source plate into the dispensing device. Loading distribution system800 can further comprise an incubator adapted to store the source plate,for example, to keep it in a cooler and more humid environment relativeto the immediately surrounding atmosphere. Loading distribution system800 can comprise a source plate-handling device adapted to translate asource plate from the incubator to the dispensing station. The incubatorcan comprise a de-lidder adapted to remove a lid from a source plate inloading distribution system 800. The de-lidder in loading distributionsystem 800 can further be adapted to place a lid on a source plate.

In some embodiments, when carriage 874 is not positioned beneathdispensing device 814, a source plate and wash pallet 864 can bepositioned under dispensing device 814. As illustrated in FIG. 91,source plate and wash pallet 864 can comprise a washing tray 861 and asource plate holder 863. Source plate-handling device 822 can pick-upand deposit a source plate 862 from source plate holder 863 using agripper 823. Source plate 862 can be covered using a lid 860. Lid 860can be placed on source plate 862 by a de-lidder 858. De-lidder 858 cancomprise a lifting device 856 adapted to lift and hold lid 860. Sourceplate and wash pallet 864 can be disposed on an elevator mechanism (notillustrated) to move source plate and wash pallet 864 within range ofdispensers 868. Source plate and wash pallet 864 can be in a restposition or a washing position. While in a rest position, washing tray861 can be covered using a dust cover 866. Dust cover 866 can be hinged.In some embodiments, loading distribution system 800 can furthercomprise a plurality of source plates in the incubator, wherein thedispensing device comprises a plurality of multi-tip dispensing heads,and the source plate handling device can be adapted to translate one ormore of the plurality of source plates from the incubator to each of theplurality of multi-tip dispensing heads.

In FIG. 66( b), a washing tray can be disposed on a washing tray pallet865′ adapted to elevate the washing tray under dispensers 868′ of adispensing device 814′. A source plate 862′ can be disposed on a sourceplate pallet 864′ that can be positioned under dispensing device 814′.Source plate-handling device 822′ can comprise dual end effectors topick-up and deposit a source plate 862′ on source plate pallet 864′.

As illustrated in FIGS. 68( a)-(c), source plate and wash pallet 864 cancomprise washing tray 861 and holding source plate 862. As illustratedin FIGS. 68( a)-(c) a dispensing device can comprise 96-fixeddispensers. FIG. 68( a) illustrates an internal dispenser wash.Dispensers 868 can be immersed in a fluid disposed in internal washslots 878. FIG. 68( b) illustrates an external dispenser wash.Dispensers 868 can be immersed in a fluid disposed in external washslots 876. FIG. 68( c) illustrates aspiration by dispensers 868. Theillustration depicts 96-dipsensers into a 384-well source plate. Eachrespective dispenser can be illustrated disposed in every other wellalong every row and every column. In some embodiments, each dispensingdevice can be adapted to be loaded by aspirating fluid from a fluidsource. The fluid source can be disposed in loading distribution system800, for example, in the storage unit or in a separate, second storageunit. Each storage unit can comprise an incubator.

As illustrated in FIG. 69, a ceiling mounted plate-handling device 830can be adapted to retrieve microplate 20 from a plate storage unit 828.Plate-handling device 830 can pick-up and remove microplate 20 from atable 872. Table 872 can be moved along a conveyer 802. The ceilingmount configuration can provide for an unobstructed range of motion byplate-handling device 830. The ceiling mount configuration can provideclearance for an arm of plate-handling device 830. Plate storage unit828 can be adapted to translate racks of microplates allowingplate-handling device 830 to access microplates 20 stacked in each rackof plate storage unit 828. Plate storage unit 828 can provideenvironmental control. Plate storage unit 828 can be designed formobility. Plate storage unit 828 can be designed for off-line operatorloading and unloading. Microplates 20 can be stored in plate storageunit 828 in a landscape orientation with respect to conveyer 802.Microplates 20 can be stored in plate storage unit 828 in a portraitorientation with respect to conveyer 802.

In some embodiments, an interval required to unload and reload amicroplate from loading distribution system 800 can be a rate-limitingfactor when determining throughput of loading distribution system 800. Aplate gripper, automated and robotic, in combination with a carriageadapted to allow simultaneous or substantially simultaneous, unloadingand reloading of microplates on the carriage, in a minimum amount oftime, can be provided.

Referring now to FIG. 70, a carriage 874 comprising a table 872 isillustrated. Microplate 20 can be disposed on table 872. Carriage 874can comprise locating pins 882 a, 882 b, and 882 c disposed on table872. A ratchet 888 can be disposed on table 872. As illustrated in FIG.72, ratchet 888 can be spring-loaded by a spring 910. When microplate 20is disposed on table 872, spring 910 can secure microplate 20 againstlocating pins 882 a, 882 b, and 882 c. Spring 910 can be automated.Spring 910 can be actuated and/or released by a manufacturing controlsystem. Spring 910 can be used to position microplate 20 on table 872,allowing stations disposed along conveyer 902 to be correctly oriented.A self-conveyance device 909 can propel carriage 874 around conveyer 802(not illustrated). In some embodiments, loading distribution system 800can further comprise a conveyer on which or with which the table and/orthe alignment stage can be moved or translated. Loading distributionsystem 800 can comprise a carriage, for example, that can ride on,along, and/or with the conveyer. The carriage can be adapted to betranslated to one or more of the plurality of processing stations. Thecarriage can be adapted to translate the table along the conveyer to oneor more of the plurality of processing stations.

According to some embodiments, table 872 can comprise a plurality oftables and the carriage can comprise a plurality of carriages eachrespectively adapted to translate one or more of the plurality oftables. Each carriage can comprise a self-conveyance device, forexample, a translation motor or servomotor, and the plurality ofcarriages can be disposed on or along a conveyer. In some embodiments,each of the plurality of carriages can comprise a plurality of automatedactuators and a self-conveyance device, for example, wherein theself-conveyance device can comprise a conduit for transferring controlsignals to the plurality of automated actuators. The conveyer cancomprise a track, for example, in the form of a circle, oval, or otherloop. The loop can be endless.

In some embodiments, loading distribution system 800 can be adapted toconvey the table along the X-axis direction. The conveyance can berepeatably positionable to within about 100 micrometers of a predefinedlocation. A conveyer can be used that serially translates one or more ofa plurality of tables, for example, with each table being disposed on arespective carriage. The plurality of tables can be translated, forexample, consecutively translated, to each of the plurality ofprocessing stations.

In some embodiments, a vacuum line supply 890 can provide communicationfrom table 872 to a bellows 896. Bellows 896 can communicate with avacuum connection shoe 907.

In some embodiments, carriage 874 can comprise a mechanism to lift orraise a first microplate, allowing a second microplate to be placedunder the first microplate. Carriage 874 that transports microplate 20between stations of loading distribution system 800 can comprise a setof grippers comprising a first cam 884 and a second cam 886, which canhold up microplate 20 without microplate 20 resting on table 872 ofcarriage 874. First cam 884 and second cam 886 can be pivotally attachedto self-conveyance device 909. Table 872 of carriage 874 can move up anddown vertically. The normal resting position of table 872 can be at amidpoint of travel for table 872, rather than a bottom point of travelfor table 872. Table 872 normally rests on a spring plunger 902 via apin 898. Table 872 can be lifted off spring plunger 902 for an upwardmotion. Table 872 can be forced down, in a downward motion, and depresspin 892 into spring plunger 902. The downward motion can allow first cam884 and second cam 886 to grab microplate 20 on table 872 and liftmicroplate 20 up off a surface of table 872.

In some embodiments, rollers 894 and 892 can be attached to first cam884 and second cam 886, respectively. A tripod 901 can be disposed in alinear bearing 904. Linear bearing 904 can be disposed vertically. Atravel of tripod 901 can raise and/or lower table 872. A roller 906 canbe attached to tripod 901.

FIG. 71 illustrates a spring 908 that holds table 872 of carriage 874against one corner.

FIG. 73 illustrates a sectioned view of spring plunger 902 that holdstable 872 (not illustrated) at an intermediate position in the Z-axis.Table 872 can be lifted off pin 898 to raise table 872 for dispensing orspring 912 can be overpowered to depress table 872 for microplateswapping operation as described herein.

FIG. 74 is a perspective view illustrating an embodiment of a pressuresource 918 adapted to communicate with vacuum connection shoe 907.Vacuum connection shoe 907 can comprise a port 920 on the opposite sidethat can engage with a vacuum supply port 916 disposed in a frame 914attached to conveyer 902. Bellows 896, or other means known in the art,can allow a flexible connection between vacuum connection shoe 907 andtable 872 that can move up and down, and shift sideways.

In FIG. 74, vacuum connection shoe 907 can be disposed next to vacuumport 916 on frame 914. When a carriage is at a station, for example, aloading station, or a dispensing device station, a valve (notillustrated) opens where vacuum port 916 is disposed on frame 914. Avacuum retainment valve (not illustrated) can be disposed on carriage874 along bellow 896 or vacuum line supply 890.

In some embodiments, vacuum connection shoe 907 can be elongated so thata vacuum connection is established before table 872 can reach the stopposition at a station. This elongated vacuum connection shoe can make asignificant difference in cycle time, as a final deceleration prior tostopping a carriage at a station can be a large part of total transittime for a carriage.

FIGS. 75 and 76 illustrate cam rails 922, 924 and a slotted rail 926comprising a slot 930 for vertical motion of first cam 884 and secondcam 886 and tripod 901, respectively. Cam rails 922, 924 can be attachedto conveyer 802. Cam rails 922, 924 can control the timing of first cam884 and second cam 886 when performing a grip operation. Slotted rail926 can control a drop operation of table 872. The two operations canoccur automatically during the motion of carriage 874. The twooperations can occur simultaneously or substantially simultaneously.Carriage 874 transfer speed can take into consideration a use of camrails 922, 924 and slotted rail 926. First cam 884 and second cam 886can be fixed to carriage 874. When a station, for example, a dispensingdevice station, needs a final registration of microplate 20, table 872can float relative to carriage 874. Table 872 need not float relative tocarriage 874 at some stations, for example, a load station or an unloadstation.

Slotted rail 926 that controls the Z-axis movement of table 872 can befixed to conveyer 802. Cam rails 922, 924 can be mounted to anair-operated slide 921. Air-operated slide 921 can be attached toslotted rail 926. When carriage 874 approaches cam rails 922, 924, table872 can be floating at a midpoint, and first cam 884 and second cam 886can be open. Cam rails 922, 924 can be elevated when carriage 874approaches a station. Cam rails 922, 924 can be rising up, for example,by activating air-operated glide 921, to meet carriage 874 as it entersa station as long as cam rails 922, 924 are in position when roller 906,a Z-axis control roller, engages with slotted rail 926. When roller 906enters slot 930, tripod 901 can drop. As table 872 rests on tripod 901,table 872 can drop down with tripod 901. Prior to dropping tripod 901,rollers 894 and 892 can engage cam rails 922, 924. As rollers 894 and892 rise on a ramp of cam rails 922, 924, first cam 884 and second cam886 attached to rollers 894 and 892, respectively, close and gripmicroplate 20. As a ramp of cam rails 922, 924 continues to rise, firstcam 884 and second cam 886 can lift microplate 20 off table 872. When arelease of a gripped microplate is desired, first cam 884 and second cam886 can be dropped, by lowering air-operated slide 921 that in turnlowers cam rails 922, 924. The lowering of cam rails 922, 924 candisengage rollers 894 and 892 from cam rails 922, 924, which in turn canopen first cam 884 and second cam 886 releasing a gripped microplate 20.The release can performed when, for example, a plate gripper robot 784is ready to remove a microplate. Plate gripper robot 784 is illustratedin FIGS. 82-90 described below.

FIG. 77 is a perspective view illustrating an embodiment of a loadingdistribution system comprising carriage 874, table 872, and an alignmentstage 932. Alignment stage 932 can be disposed under a dispensing devicemount 931. A dispensing device (not illustrated) can be attached todispensing device mount 930. Table 872 of carriage 874 can engage withalignment stage 932 when carriage 874 lifts. A set of actuators 934, 936engages with three points on table 872 after carriage 874 enters adispensing station and table 872 has been raised. Alignment stage 932can comprise a long stroke actuator 935 for the X-axis since microplate20 disposed on table 872 can index over a substantial distance for somekinds of dispensing, for example, dispensing of fluids for FocusedGenome dispensing. The X-axis carries two short stroke Y-axis actuators934, 936. The Y-axis actuators 934, 936 can operate independently fromeach other to compensate for skew.

In some embodiments, loading distribution system 800 can comprise thetable, the alignment stage, and a plurality of processing stations. Thetable can be configured to engage at least one of a plurality ofmicroplates and be movable at least in an X-axis direction. The tablecan be moved together with a carriage that in-turn can be adapted tomove in the X-axis direction. The an alignment stage can be configuredto move the table and/or carriage at least in a Y-axis direction thatdiffers from the X-axis direction, for example, that can beperpendicular or at least substantially perpendicular, to the X-axisdirection. In some embodiments, substantially perpendicular can meanwithin about 15 degrees of being perpendicular. The plurality ofprocessing stations can comprise at least one or more dispensingstations and a plate-handling station. Each of the one or moredispensing stations can comprise a dispensing device adapted to dispensefluid into or onto one or more of a plurality of microplates. Theplate-handling station can comprise a plate-handling device. Theplate-handling device can be adapted to selectively pick up and depositon the table individual microplates from a plurality of microplates, atleast one at a time. In an exemplary embodiment, loading distributionsystem 800 can further comprise a microplate disposed on the table,wherein the dispensing device comprises at least 24 or more dispensers,and the microplate comprises 768 or more wells, for example, 96 or 384dispensers and 6,144 wells.

In some embodiments, alignment stage 932 works in cooperation withlocating pins 882 a, 882 b, and 882 c. A location of microplate 20 canbe offset in varying degrees from the center of dispensing device 814 tosatisfy a need to interleave subsets of dot patterns or dispensinglocations, and to form stripe pattern offsets for Focused Genomedispensing. A system requiring operator intervention to mechanicallyalign dispensing device 814 with the independent axes of motion, forexample, X, Y, and Z-axis, can be very difficult to maintain. In someembodiments, loading distribution system 800 can work without a need forprecision alignment by an operator after maintenance on loadingdistribution system 800 has been performed. Alignment stage 932 can beenhanced with a vision system based adaptive alignment system. A camera(not illustrated) can form an image of microplate 20. The image can beprocessed to derive X, Y, and/or Z movement specifications for alignmentstage 932. Table 872 can comprise reference markings (not illustrated)to determine offsets needed to compute the movement specifications.

FIG. 78 is a perspective view illustrating an embodiment of a liftingstage 940 adapted to lift carriage 874 in the Z-axis. A motorized slide938 moves a block 941 with a slot in block 941, lifting carriage 874 upand down. Roller 906 that controls the Z-axis engages with a slot inblock 941 to move table 872 of carriage 874 up for dispensing. Liftingstage 940 can be disposed in a position underneath a dispensing deviceto allow a Z-direction movement of carriage 874.

FIG. 79( a) and FIG. 79( b) are perspective views illustrating twovisual inspection station, according to some embodiments. The visualinspection stations can provide an ability to compensate for a largenumber of potential errors, assist in quality control, and alignment ofmicroplates.

FIG. 79( a) illustrates a full scan vision station disposed on conveyer802. The full scan vision station can perform a full scan of microplate20 disposed of table 872. A camera mount 941 can extend from conveyer802 to position a camera 947 over microplate 20 as it moves aroundconveyer 802. A carriage alignment device 945 can engage and properlyalign table 872 with camera 947. Carriage alignment device 945 can be amechanical device to push table 872 into a fixed position by contactingthree points on a perimeter of table 872. This can eliminate servoerrors to provide a consistent reference measurement. Carriage alignmentdevice 945 can retract from above conveyer 802, thus disengaging table872 from the full scan vision station. Carriage 874 can be docked at astation where camera 947 takes a picture of a fluid pattern deposited onmicroplate 20. The full scan vision station can provide quality control.The full scan vision station can be used to provide measurements toalignment system 932. The full scan vision station can be downstream ofthe dispensing devices for quality control of microplate 20.

A periphery scan vision system or plate check vision system can bedisposed upstream of a dispensing device to check the position andaccuracy of microplate 20, prior to a dispensing by a dispensing device.The periphery scan vision system can utilize a camera mount 941 to holdtwo cameras 946, 948. Cameras 946, 948 can be narrow focus cameras.Cameras 946, 948 can check the location of two or three dispensinglocations. The periphery scan vision system can comprise a carriagealignment 944 similar in functionality to carriage alignment device 945described above. The periphery scan vision system can comprise a markerindicia reader station.

In some embodiments, a reference microplate can be disposed on table932. The reference microplate can comprise an accurately machinedmicroplate mimicking a microplate. The reference microplate can comprisea pattern of etched dots or location that matches the desired pattern onmicroplates to be manufactured.

In some embodiments, a test target microplate can be disposed on table932. Flat blank plates can be used for making test patterns of dots. Thetest target microplate can comprise, for example, a plastic material ora cardboard material. The test target microplate does not need tocomprise wells. The test target microplate can comprise a surfaceproviding good contrast with the dot pattern. The surface can comprise acoating that can change color when liquid contacts the coating.

In some embodiments, the following sequence of operations can be usedadjust loading distribution system 800. The reference microplate can beplaced on a first carriage and the first carriage can be moved to thefull scan vision system. The dot pattern on the reference microplate canteach the camera of the full scan vision station, the desired dotlocations. Next, a test target microplate can be placed on a secondcarriage. The second carriage can be moved under a dispensing device.The alignment stage can move the table of the second carriage to theposition that the alignment stage guesses to be the correct position.The guess can be based on previous runs. A single test target microplatecan be used for one or more of the dispensing devices since the patternsfrom the individual dispensing stations can be disposed far enough apartso that they do not overlap. Lastly, the second carriage with the testtarget microplate can be moved to the full scan vision system and thedot pattern of the test target microplate can be compared to the storedmemory of the desired pattern. Offsets can be computed to adjust theposition of the alignment stages for the next cycle.

The above process can be repeated by running another test targetmicroplate through loading distribution system 800 to verify the resultsof the previous run, until achieving a desired or satisfactory run. Theabove process need not be repeated. When it is determined that the dotpattern from a particular dispensing device does not or cannot fitted toa desired pattern by adjusting the X, Y and rotary axes, then aiming ofdispensers of the dispensing device can be checked and adjusted, ifdesired. Loading distribution system 800 can alert an operator or it candevise another offset for the off-target dispenser or a subset of theoff-target dispensers. The alignment stage can move the table to oneposition and fire one set of dispensers. The alignment stage can thenmake a slight adjustment of the alignment of the table and thedispensing device, and fire another dispenser or set of dispensers. Thealignment can be dynamic while loading distribution system 800 can bedispensing fluids to the microplates. The slight penalty of a microplatethat fails quality control and/or a slight increase in the overall cycletime can be preferable to stopping loading distribution system 800 formaintenance. This process can be useful for expediting, for example,small orders of custom microplates.

In some embodiments, once loading distribution system 800 adjusts for aproduction operation, a microplate can be loaded onto a carriage. Thecarriage can be moved to the periphery scan vision system. The locationof two or more wells can be checked and a new offset for this carriageand microplate set can be added to loading distribution system 800offsets. This new offset can adjust for variations in carriages,variations in how a microplate is placed on a carriage, and moldingvariations in the microplates. If the dispensing locations wells are toofar or too close to each other or to the edge of the microplate, themicroplate can be rejected and the microplate need not be spotted. Ifthe well spacing is within limits but substantially off from the ideal,the error can tend to be cumulative rather than random. This means thateach dispensing location can be almost perfectly spaced relative toadjacent dispensing locations, but that this spacing can be alwaysslightly larger or smaller than specification. This can imply that thefarthest dispensing locations on the microplate can be out ofspecification in relation to each other. Loading distribution system 800can divide the microplate into halves or quadrants, compute an offsetfor each quadrant, and then dispense to each quadrant with a respectiveoffset.

According to some embodiments, a fluid distribution system can comprise:a table configured to engage at least one of a plurality of microplatesand movable at least in an X-axis direction and in a Y-axis directionthat differs from the X-axis direction; a dispensing device adapted todispense fluid into or onto one or more of a plurality of microplates; aplate-handling station comprising a plate-handling device adapted toselectively pick-up microplates from and deposit microplates on thetable; an inspection station adapted to image a microplate when amicroplate is disposed on the table; a calculating device adapted tocompute offsets that can comprise at least an X-axis direction offsetand a Y-axis direction offset, based on an image provided by theinspection station; and a control device adapted to control anadjustment of a relative position of the table based on offsets computedby the calculating device.

According to some embodiments, the calculating device can be adapted tocompute positions of at least two dispensing locations on a microplatefrom an image of the microplate. The calculating device can reject amicroplate if the computed positions are not within a predeterminedspecification. The calculating device can be adapted to divide the imageinto portions and compute positions of at least two dispensing locationsin each image portion. The calculating device can reject a microplate ifrespective computed positions of an image portion are not within atleast one predetermined specification. The control device can be adaptedto control movement of the table with the respective offset for eachimage portion being dispensed to by the dispensing station. Themicroplate can comprise a reference target plate.

According to some embodiments, the system can comprise a marking indiciareader such as marking indicia reader 804 adapted to read a markingindicia disposed on a microplate when a microplate is disposed on thetable. The system can comprise a memory or storage device capable ofstoring offsets indexed by the marking indicia for one or more of aplurality of microplates. The system can comprise an alignment stageconfigured to move the table in the X-axis direction and in the Y-axisdirection.

According to some embodiments, the calculating device can computeoffsets. Either retrieving from the storage device offsets indexed to arespective marking indicia, or computing and saving into the storagedevice offsets indexed by the respective marking indicia, for one ormore of a plurality of microplates.

According to some embodiments, the table can comprise a plurality oftables and each table can comprise a respective table identifier. Thestorage device can store offsets by the table identifier and markingindicia pair. The computing device can retrieve offsets by the tableidentifier and marking indicia pair.

According to some embodiments, the system can comprise a quality controlinspection device adapted to inspect an image of two or more dispensingsonto a microplate. The quality control inspection device can be adaptedto reject a microplate if an image of two or more dispensings is notwithin at least one predetermined specification. The quality controlinspection device can be adapted to compute dispensing station offsetsthat can comprise at least an X-axis direction offset and a Y-axisdirection offset, based on the image.

According to some embodiments, the quality control inspection device canbe adapted to inspect an image of a microplate. The quality controlinspection device can be adapted to divide the image into portions. Thequality control inspection device can be adapted to compute positions oftwo or more dispensings in each image portion. The quality controlinspection device can be adapted to reject a microplate if positions foreach image portion of the microplate are not within at least onepredetermined specification. The quality control inspection device canbe adapted to adjust a dispenser of a dispensing device if positions andvolumes for each image portion of the microplate are not within at leastone predetermined specification. The microplate can comprise a testtarget microplate.

In some embodiments, loading distribution system 800 can be useddispense dry beads. Loading distribution system 800 can use dry beadsrather than fluids to deposit probes. The dry dispensing can face thesame issues of how to align a series of interleaved dispensing devices.Dropping dry beads on a test microplate does not provide a useful testpattern. The individual dispensing devices can comprise ink jet heads orsharp pins that can be machined in a fixed pattern relative to the beadoutlet points. A test microplate can be run through loading distributionsystem 800 and the jets or pins can be activated to create a visible dotpattern that can be checked by a vision system.

FIG. 80 is a top-plan view illustrating table 872 comprising a vacuumtrench 954 and a gasket 956. When a microplate is disposed on table 872,a pressure source (not illustrated) can be connected to a vacuum inlet952, to form a vacuum between a surface of microplate 20 and table 972.FIG. 74 illustrates an embodiment of a pressure source communicatingwith table 872. FIG. 80 illustrates an embodiment of table 872comprising four locating pins and no ratchet, in contrast to table 872of FIG. 70.

In some embodiments, a table can provide for initial microplateregistration to a carriage at a load station. Vacuum formed between amicroplate surface and a table can be used to flatten a microplate. Thevacuum can also hold a microplate in place for a dispensing operation.Loading distribution system 800 can operate under a tight tolerancewindow. A dispensing device and a microplate can be aligned by variousdevices described to be within, for example, about 100 μm, about 40 μm,or within about 10 μm. These tolerances can allow dispensing intomicroplates, for example, high-density microplates. The alignmentdevices can be supplemented with vision and/or laser based activealignment systems, for additional accuracy if desired. Alignment to thetight tolerances can compensate for potential molding errors, headalignment errors, track variability, and table on carriage errors.

FIG. 81 is a perspective view illustrating a dispensing device 814including a plurality of dispensers 868.

FIGS. 82-84 are perspective views illustrating plate gripper robot 784.Plate gripper robot 784 can comprise a pair of jaws—a lower jaw 786 andan upper jaw 788. Upper jaw 788 can be mounted above lower jaw 786.Plate gripper robot 784 can include actuators 784 and 790 to pivotallymove an upper jaw-clamping portion 788 a and a lower jaw-clampingportion 786 a, respectively.

In some embodiments, as illustrated in FIG. 85 lower jaw 786 can bring afirst microplate 20 d to table 872 and can place first microplate 20 don table 872 under a second microplate 20 c that carriage 874 can beholding above table 872 using first cam 884 and second cam 886. Asillustrated in FIG. 86, plate gripper robot 784 can release firstmicroplate 20 d from lower jaw 786, placing first microplate 20 d ontable 872. As illustrated in FIG. 87, first cam 884 and second cam 886can release, and upper jaw 788 can grab second microplate 20 c. Firstcam 884 and second cam 886 can release second microplate 20 c asdescribed in FIG. 75 and FIG. 76.

FIG. 88 illustrates plate gripper robot 784 removing second microplate20 c from table 872. As illustrated in FIG. 89, plate gripper robot 784can transfer second microplate 20 c to plate storage unit 828. At platestorage unit 828, plate gripper robot 784 can place second microplate 20c on an empty shelf. The next lower shelf in plate storage unit 828 canbe empty to provide clearance for lower jaw 786.

As seen in FIG. 90, lower jaw 786 grasps a third microplate 20 e on fromplate storage unit 828 without plate gripper robot 784 needing to shiftto another position. Third microplate 20 e can now be treated as firstmicroplate 20 c of FIG. 85 and the process can be repeated again.

In some embodiments, after a stack in plate storage unit 828 has beenprocessed, plate gripper robot 784 can shift two microplates from thetop of the stack to the bottom of the stack. This can provide emptyspaces for the process, and can allow the process to repeat during anext pass. In some embodiments, the table can comprise a plate gripper.The plate gripper can be adapted to grip and/or, lift to an elevatedposition, a first microplate. Starting with a first microplate disposedon the table, the plate-handling device can be adapted to lift the firstmicroplate and deposit a second microplate underneath the firstmicroplate while the first microplate is in the elevated position.Loading distribution system 800 can comprise a plate gripper releasedevice that can be adapted to release the plate gripper from grippingthe first microplate. The plate gripper release device can enable theremoval of a first microplate from the plate gripper.

Even further details regarding various other uses and configurations ofthe plate gripper and systems using the same can be found in U.S. PatentApplication entitled “Dual Nest Microplate Spotter” to Lehto, filed thesame day as the present application.

In some embodiments, a plate gripper robot can approach a table with anew microplate. The plate gripper robot can dispose the new microplateon the table. The plate gripper robot can grip the top microplate. Theplate gripper robot can then release the new or bottom microplate. Theplate gripper robot can then remove the top microplate. At the platestorage unit, the plate gripper can place the microplate in its top jawson an empty shelf. There can be two empty adjacent shelves in a hotel,for example, the top empty shelf can receive a microplate, and the nextempty shelf can be unused, for example, for gripper clearance. The shelfbelow the two empty shelves can hold a next microplate. The lower jawsof the plate gripper robot can than grab a microplate from the shelfholding the next microplate without needing to shift to another positionalong the plate storage unit. The cycle can then be repeated to (1)place a microplate gripped by the lower jaws on the table, (2) grip andremove a microplate raised above the table using the upper jaws, (3)return the microplate in the upper jaws to the plate storage unit, and(4) grab a microplate in the lower jaw from the next shelf holding amicroplate. In some embodiments, the plate-handling device in loadingdistribution system 800 can comprise a two-jaw plate gripper device. Thetwo jaws can be positioned one over the other. Each jaw can be adaptedto grip a microplate. The plate-handling device can be adapted to gripand remove a first microplate from the table and substantiallysimultaneously deposit a second microplate on the table.

In some embodiments, a carriage or pallet can move microplates along aconveyer in a portrait orientation. It can be desirable to include asmany of the carriage functions as possible off board of the carriage fordesign simplicity. In some embodiments, a register plate function can beoff carriage. A vacuum pallet function applied to chuck can be oncarriage. A Z-motion can be off carriage. A Y-motion can be offcarriage. A vacuum sensor can be off carriage. A register sensor can beoff carriage. A bar code reader can be off carriage. A Docking, Commandand Data Acquisition (CDA), signal, and power function can be providedon a carriage. In some embodiments, loading distribution system 800 cancomprise a lift. The lift can be configured to move the table in aZ-axis direction. The Z-axis direction can be different from both theX-axis direction and the Y-axis direction. The Z-axis direction can be,for example, perpendicular or substantially perpendicular, to both theX-axis direction and the Y-axis direction. In some embodiments,substantially perpendicular can mean within about 15 degrees of beingperpendicular.

In some embodiments, the microplate can be pushed at a corner while on aload station of the conveyer. A vacuum chuck can be onboard everycarriage. A Z-motion actuator can be disposed beneath the carriage. Thiscan provide clearance and can move the vacuum chuck up to meet adispensing device. A Y-motion actuator can reside outside of thecarriage. The actuator can utilize a ram to drive a table to a referencelocation. A vacuum sensor can be disposed on the vacuum line supplyproximate a carriage docking mechanism. A register sensor-can determinecorrect microplate placement, for example, by checking a pressure on thevacuum line supply. A machine indicia reader, for example, a bar codereader, can be used with a mirror to reflect a bar code on a microplateto separate reader assembly. In some embodiments, 50-micronrepeatability can be desired for X, Y, and Z direction movements at adispensing station. The carriage can be driven on a conveyer or track bya linear stepper motor. The dispensing device and dispensers therein canbe held stationary. Various components, for example, the conveyer, ofloading distribution system 800 can be provided with EMI shielding.

FIG. 91 is a perspective view illustrating source plate and wash pallet864 comprising washing tray 861 and source plate holder 863. A sourceplate 862 can be disposed in source plate holder 863. Washing tray 861can comprise internal wash slots 878 and external wash slots 876.Washing tray 861 can be available from Aurora Discovery, Inc.

Source plate-handling device 822 can pick-up and deposit a source plate862 from source plate holder 863 using a gripper 823. Source plate 862can be covered using a lid 860. Lid 860 can be placed on source plate862 by a de-lidding device 868. De-lidding device 868 can comprise alifting device 856 adapted to lift and hold lid 860. Source plate andwash pallet 864 can be disposed on an elevator mechanism (notillustrated) to move source plate and wash pallet 864 within range ofdispensers 868. Source plate and wash station 814 a can be in a restposition or a washing position, when an elevator mechanism is used.While in a rest position, washing tray 861 can be covered using a dustcover 866. Dust cover 866 can be hinged.

FIG. 92 is a perspective view illustrating a source plate and washstation 814 a comprising at least one source plate and wash pallet 864.This embodiment of source plate and wash station 814 a can service twodispensing stations simultaneously or substantially simultaneously.Washing tray 861 and source plate holder 863 can be placed next to eachother on a platform or source plate and wash pallet 864. Source plateand wash pallet 864 can be disposed on a first slide 867. Vacuum cups856 can grab and hold lid 860, a standard plate cover. Dust cover 866can cover washing tray 861. A support 858 can be used to hold vacuumcups 856. Source plate and wash pallet 864 can normally wait in aposition that presses washing tray 861 and source plate 862 up againsttheir respective lids. Washing tray 861 can be covered by dust cover 866that can be permanently attached to a frame. FIG. 98 is a side-plan viewof source plate and wash station 814 a in a wait position with respectto conveyer 802 and dispensing device 814.

As illustrated in FIG. 93, if source plate and wash station 814 a can beextended to aspirate a dispensing device from source plate 862, thensource plate and wash pallet 864 drops and vacuum cups 856 retain lid860.

As illustrated in FIG. 94, if source plate and wash station 814 a isgoing to extend to wash dispensers of a dispensing stations, vacuum cups856 do not turn on and lid 860 stays with source plate 872. FIG. 99 is aside-plan view of source plate and wash station 814 a in the washposition with respect to conveyer 802 and dispensing device 814.

As illustrated in FIG. 95, to swap source plate 872 out with a freshsource plate from source plate storage unit 826, a second slide 869stays retracted. First slide 867 slides crossways, and shifts toone-side so that source plate 872 is not under lid 860 holding mechanismand an external SCARA or 5-axis robot, like store plate-handling unit822 can load and unload the source plate 872.

As illustrated in FIG. 96, source plate and wash station 814 a canextend on second slide 869 to position source plate 862 for aspirationby a dispensing device.

As illustrated in FIG. 97, source plate and wash station 814 a canextend on first slide 867 and second slide 869 to position washing tray861 to wash dispensers.

In some embodiments, for a wash operation carriages can be stopped alongthe conveyer at locations away from the dispensing devices to allowclearance of a washing tray moving mechanism. The moving mechanism cantravel along a fixed linear track that can bring the washing tray to theconveyer. Initially, the washing tray can be located beneath a fixedcover plate that can include an embedded seal surface that the edges ofthe washing tray can seal against when the bath is in the up or waitposition under the fixed cover. The washing tray can be lowered slightlyin the Z-direction to unseal the washing tray. The washing tray can thenmove along a linear track towards the conveyer. When the washing tray isclear of the fixed cover, the washing tray can be raised to present thewashing tray to the dispensers of a dispensing station. The washing traycan move down and can index in the Y-direction to accomplish bothinternal and external tip washing operations. When a wash cycle iscomplete, the tray can move down and back towards the rest positionalong the linear track.

In some embodiments, for an aspirate operation, a robot arm can remove acorrect source plate from an incubator and place it onto a source platelocation. The source plate can be moved to a de-lidder that can bemounted under a dust cover. The lid of the source plate can be removedusing the de-lidder.

FIG. 100 is a perspective view illustrating a hotel and a movable entryguide. In some embodiments, reliable insertion of microplates intoshelves can be facilitated by adding an entry guide 974 that captures aleading edge of a microplate. The vertical position of the edge can varyfrom microplate warping and/or variation in how a microplate can begripped by a jaw of a plate gripper robot. A shelf 970 can providesupport for plate storage unit 828. Entry guide 974 can be indexed usinga linear motor 972.

FIG. 101 is a process flow diagram illustrating a software command andcontrol architecture for a loading distribution system, according tosome embodiments. A system controller 982 can networked to an enterpriseresource planning (ERP) system 983, using an inter or intra network 985.ERP system 983 can provide work order requests to system controller.

In some embodiments, system controller 982 (FIG. 101) can manage andtrack source plates and microplates at various locations in loadingdistribution system 800 (FIGS. 64 and 65). Locations for a source platecan comprise, for example, in a source plate storage unit like anincubator, in one or more source plate holders, or in one or moregrippers of one or more source plate handling devices. Locations for amicroplate can comprise, for example, in one or more plate storageunits, in or on one or more tables, or in one or more jaws of one ormore plate handling devices. System controller 982 can be adapted totrack and trace the contents of one or more dispensers, each disposed inone or more respective dispensing devices.

When processing a work order or manufacturing microplates, systemcontroller 982 provides control, control, and communication for washstation assemblies module 984, a tip firing controller 986, a dispensingassemblies module 988, an incubator controller 990 also known as asource storage unit controller, an incubator robot controller 992 alsoknown as a storage plate handling device controller, a fluidicscontroller 994, a hotel module 996 also known as a storage unitcontroller, a hotel robot controller 998 also known as a plate handlingdevice controller, a bar code controller 976 also known as a markingindicia reader controller, a XYZ motion controller 978, and a qualitycontrol controller 929. Wash station assemblies module 984, tip firingcontroller 986, dispensing assemblies module 988, incubator controller990, incubator robot controller 992, fluidics controller 994, hotelmodule 996, hotel robot controller 998, and bar code controller 976 canbe provided as part of one or more Original Equipment Manufacturer (OEM)packages including Application Protocol Interfaces (API) for allsubassemblies. System controller 982 and XYZ motion controller 978 canbe provided using real-time manufacturing protocols, for example,Supervisory Control And Data Acquisition (SCADA), a computer system forgathering and analyzing real time data. Quality control controller 929can comprise a decision maker. QC controller 929 can gather data andstatus from various systems comprising a loading distribution system, torender a decision for each microplate processed by loading distributionsystem.

In some embodiments, the array of dispensers can be aligned to amicroplate, in order to accomplish parallel dispensing of differentreagents into different locations at the same time. Dispensers candispense spots of an assay reagent into one or more locations of amicroplate by, for example, aspirating a volume of assay reagentsufficient for multiple spots. The aspirated volume can subsequently bedispersed as spots into multiple locations, where each location receivessubstantially the same mass of assay reagent.

A dilution problem can be observed using arrayed dispensers. Dilutioncan occur because a dispenser system fluid can dilute an assay reagent,as it is dispensed. Because a dispenser can dispense a volume of thereagent and system fluid, a reduced mass of assay reagent can bedeposited into each location from dispensing action to dispensingaction.

In some embodiments, a dispenser can be programmed to compensate for thedilution affect. The aspirate and dispense arrayed liquid handlingtechnologies, can dispense different amounts of assay reagents for eachnozzle for each dispense action. The level of dilution can be measured,and the measured curves can be used to calibrate the effect of dilution.In some embodiments, a method for calibrating the observed diffusion ona tip-by-tip basis, and compensating for the loss of dispensed assayreagent per nozzle from dilution by programming dispensing to dispensemore solution per spot, is provided. A required increase in spot volumescan be calculated by mathematically integrating an area under afluorescence-dispense calibration curve. In some embodiments, dynamicprogramming of the dispense volumes can provide microplate to microplatereproducibility of dispensed mass of assay reagents (spots), and canreduce assay reagent waste by allowing the use of highly diluted assayreagents from the dispensing device.

In some embodiments, methods of spotting assay reagents based ondispenser arrays, into microplates, consistent with the banded format offilling devices, and the production of source plates for spotting, areprovided.

In some embodiments, assay 1000 can be distributed on microplate 20using a filling apparatus, such as filling apparatus 400, a roboticfiller, or a manual filler to distribute one or more components of assay1000 across microplate 20 in columns or bands, for example, asillustrated in FIG. 102. For microplates that accommodate more than onesample, the sample distribution can map to this columnar or bandedformat.

FIG. 102 illustrates sample distribution in a banded format using arobotic or manual filler head. The head comprises tips 746, 748, 750,752, 754, 756, 758, and 760, respectively. Tips 746, 748, 750, 752, 754,756, 758, and 760 can aspirate fluids from source plate 862. Sourceplate 862 can comprise, for example, a 96 or a 384-location plate,including, for example, biological reagents or pre-amplified samples.Tips 746, 748, 750, 752, 754, 756, 758, and 760 can distribute theaspirated samples across microplate 20 to form bands or columns acrossmicroplate 20, for example, bands about 9 mm wide, bands about 4.5 mmwide, bands about 2.25 mm wide, or bands about 1.125 mm wide. Themicroplate can include, for example, 6,144 wells. Tips 746, 748, 750,752, 754, 756, 758, and 760 can dispense individual samples in bandsacross a plurality of rows of microplate 20. As illustrated in FIG. 102,tip 746 can correspond to band 746′, tip 748 can correspond to band748′, tip 750 can correspond to band 750′, tip 752 can correspond toband 752′, tip 754 can correspond to band 754′, tip 756 can correspondto band 756′, tip 758 can correspond to band 758′, tip 760 cancorrespond to band 760′, and tip 762 can correspond to band 762′. In anexemplary embodiment, tip 746 can load an eight-row column that is atotal of 9 mm wide, from one end to the other end of the card, toinclude band 746′ illustrated in FIG. 102. With a number of sweeps alongthe card, back-and-forth, a band of sample can be loaded into themicroplate, and with an 8-tip dispenser, the entire 6144 wells of a 6144well microplate can be loaded with eight motions of the filler toachieve loading one respective well at a time, for each dispenser tip.

FIG. 31 illustrates the use of a dead row between sample-loaded wellsthat can be used to avoid cross-contamination of two rows to be tested,taking advantage of a banded format. FIG. 103 illustrates a microplate764. In the following discussion, rows run from left to right.Microplate 764 includes three rows, illustrated from left to right inthe figure, including a first row into which a first sample is loadedand including sample wells 766. A second row into which a second sampleis loaded includes sample wells 770. The row containing sample wells768, located in between the rows respectively containing sample wells766 and sample wells 770, can be used as a dead row and can be skippedduring a sample loading process. If any of the first or second samplesmight stray from its intended row, it can be captured in the dead row.That is, if a sample deposited in well or location 766 or well orlocation 770 of microplate 764, carries over to an adjacent location768, no problem arises because the results of any assays in wells 768would not be analyzed. For example, when using a robotic or manualfiller, any possible cross-contamination between samples can beprevented by leaving approximately one unused row (a “dead row”) betweeneach band of loaded samples in the microplate. The dead row can compriseone or more rows.

In some embodiments, a method of avoiding cross-contamination of aplurality of samples disposed in locations of a microplate can beprovided. The method can include loading a filling device that caninclude a plurality of dispensers, each dispenser can include a fluid;translating the filling device along a translation path traversing amicroplate that can include rows of locations; and dispensing a band ofa respective fluid from each of the dispensers along a portion of thetranslation path to load rows of the locations, where the bands do notcontact one another and the rows include loaded rows and a dead rowbetween otherwise adjacent loaded rows.

Bands can contain the same set of samples or assay reagents across themicroplate. One row can be eliminated from each band on the microplate.Where one band or one sample is provided on the microplate, there can beno need for a dead row to prevent sample cross-contamination.

In some embodiments, the dead rows of a microplate can be left empty orcan be spotted with one or more components of assay. A buffer, forexample, a TaqMan buffer, comprising no templates in common with theassay reagents in the bands, can be used to fill locations in a deadrow. In some embodiments, each microplate can comprise an m×nconfiguration. Dead rows do not have to comprise wells or fluidlocations. Dead rows can comprise other markings or features, forexample, mold ejector pins can be disposed in the dead rows to improve arelease of the microplate from a mold. Dead row wells or locations canbe loaded with a calibrating dye or other marker or control substanceuseful in calibrating, for example, with respect to fluorescence orbackground noise. Dead row wells or locations can be loaded with a dyeor other marker useful in providing identifiable locations on themicroplate.

FIG. 104 illustrates a system according to some embodiments formanufacturing source plates and spotted microplates. Loadingdistribution system 800 can include: a plate-handling station 774 formoving at least one microplate; a first dispensing station 780 and asecond dispensing station 782; a source incubator 776; and a microplateincubator 778. Each dispense station can dispense fluid, for example,into or onto a microplate. Each dispense station can aspirate fluid fromone or more source plate. Plate-handling station 774 can move sourceplates (not illustrated) in and out of source incubators 778.Plate-handling station 774 can move and microplates in and out ofdispensing stations 780, 782. The source plates can be stored inincubators when not in use.

In some embodiments, source plates can be stored, optionally lidded, insource incubator 776 that can circulate, for example, high humidityfiltered air around the source plates. This can, for example, preventevaporation of the assay reagents. There can be a delay between whensource plates are prepared and when they are used for spottingdestination microplates. The delay can be problematic becauseevaporation can adversely change the concentration of the reagents.

In some embodiments, the spotted assay reagents can be dried and themicroplates can be protected from dust during production. Drying ofmicroplates can take place in microplate incubator 778. The destinationmicroplates can be stored, optionally lidded, in microplate incubator778 that can circulate low humidity filtered air around the microplates.Because the spotted assay reagents can be dried within microplateincubator 778, a post-batch drying step for the microplates can beeliminated. In some embodiments, loading distribution system 800 can behoused in an enclosure such that the housing can enclose loadingdistribution system 800. The housing can comprise a class 1000 orcleaner clean room.

Plate-handling station 774 can be adapted to selectively pick up anddeposit in dispensing station 780, 782, individual microplates, at leastone at a time. The plate-handling station 774 can include, for example,a robotic arm. The plate-handling station 774 can be adapted tosimultaneously remove a first microplate from an incubator and deposit asecond microplate an incubator. Dispensing stations 780 and 782 caninclude at least 96 dispensing tips, or at least 384 dispensing tips.Each dispensing station can include a plurality (two or more) ofdispensers. Dispensing stations 780 and 782 can further include aplurality of (two or more) storage reservoirs. The source incubator 776can store a source plate. The microplate incubator 778 can store amicroplate that is unspotted, partially spotted, or fully spotted. Thesource incubator 776 can include circulated high humidity filtered airin order to prevent evaporation of the source assay reagents from thestored source plate. Microplate incubator 778 can include circulated lowhumidity filtered air to dry the spotted assay reagents. Microplateincubator 778 can maintain the spotted dried assay reagents in a driedstate on the spotted microplate. Microplate incubator 778 can prevent apost-batch drying step.

The plate-handling station 774 can be adapted to selectively pick up anddeposit individual source plates from the source hotel 776, microplatesfrom the microplate hotel 778, or microplates and/or source plates fromdispensing station 780, 782. The plate-handling station can transfersource plates from the dispensing station 780 and 782 to the appropriatesource incubator 776. The plate-handling station can transfermicroplates from the dispensing station 780 and 782 to the appropriatemicroplate incubator 778. The source plates and/or the microplates canoptionally be lidded. The incubators can include a device for liddingand de-lidding a source plate.

In some embodiments, methods and systems are provided that improve themanufacturing of microplates by: increasing microplate to microplatereducibility and reducing assay reagent waste; preventing samplecross-contamination from the use of robotic and manual fillers; reducingevaporation loss of assay reagents from source plates; assisting in thedrying of spotted assay reagents on microplates, and avoiding apost-batch step of drying the microplates; and reducing dustcontamination of both source and microplates.

FIG. 105 is a top-plan view illustrating a mapping of fluid locations ofa 384-location source plate into a dispensing device comprising 96dispensers, further into a 6,144-microplate. Microplate 20 can comprisea plurality of grids, for example, 96-grids. A grid 854 can comprise 64locations. Each of the locations in a grip of microplate 20 can bedispensed into or onto by a respective dispenser 868 of dispensingdevice 814, when dispensing device 814 comprises 96-dispensers. Aquarter of a grid 852, 16 locations, illustrates a location map pattern.The locations in quarter of a grid 852 can be addresses as 1, 2, 3, and4 for a first row; 7, 8, 9, and 10 for a second row; 17, 18, 19, and 20for a third row; and 25, 26, 27, and 28 for a fourth row. Loadingdistribution system 800 can dispense into a location number 1 during afirst pass over microplate 20, location number 2 during a second passover microplate 20, and so on so forth. To accomplish this, loadingdistribution system 800 can control the X and Y placement of microplate20 using X-Y alignment, for example, as provided by alignment stage 932as described above when dispensing device 814 is fixed or stationarywith relative to microplate 20, or by offsetting each dispenser 868 ofdispensing device 814.

In some embodiments, source plate 862 can be divided into 96-grids, eachgrid 848 comprising 4-locations for fluid aspiration. Loadingdistribution system 800 can aspirate from a location number 1 during afirst pass over source plate 862, location number 2 during a second passover source plate 862, and so on so forth. To accomplish this, loadingdistribution system 800 can control the X and Y placement of sourceplate 862 using X-Y alignment, for example, as provided by source plateand wash station 814 a as described above when dispensing device 814 isfixed or stationary with relative to microplate 20, or by offsettingeach dispenser 868 of dispensing device 814 while holding source plate862 in fixed position.

In some embodiments, a system and method for manufacturing a microplatecomprising a plurality of fluid samples, for example, about 768 or moresamples, about 1536 or more fluids, about 3072 or more fluids, about6,144 or more fluids, about 12,288 or more fluids, are described. Insome embodiments the plurality of fluids can all be the same fluid andin some embodiments each fluid can be different from all the otherfluids. The plurality of fluids can reside in or on a microplate.

In some embodiments, fluids to loading distribution system 800 can beprovided using a source plate, for example, a multiwell source plate.The source plate can comprise 24 or more wells, for example, 48 or morewells, 96 or more wells, 192 or more wells, 384 or more wells, or 768 ormore wells.

In some embodiments, a dispensing device comprising a plurality ofdispensers can be used in the present teachings. The dispensers cannumber 24 or more tips, for example, 48 or more tips, 96 or more tips,192 or more tips, 384 or more tips. The dispensers can be, for example,piezo-electric spotting tips. The dispensers can be disposed in an SBSmicrotiter footprint, for example, the footprint and pitch distributionof a standard 96 well microtiter plate, a 192 well microtiter footprintpitch, a 384 well microtiter footprint, etc. In some embodiments, thedispensers can be fixed in position. In some embodiments, the dispenserscan be moveable within a subportion of the dispensing device.

According to some embodiments, a system utilizing a 384-well sourceplate using a 96-dispenser device can be used to manufacture amicroplate comprising, for example, 6,144 wells. Loading distributionsystem 800 can utilize, for example, 16, 384 well source plates, toaccess 6,144 unique fluids from the 36 times 384 or 6,144 wells. A96-dispenser device can access a 384-source plate four times, each timedrawing 96 unique fluids into corresponding 96-dispensers. Thus, thedispensing device can aspirate from a 384 well source plate 4 times.Sixteen source plates and 64 aspirations can be utilized to aspirate6,144 unique fluids. A dispenser can be positioned over a targetmicroplate comprising 6,144 wells, 64 times. For a 96 tip dispenserspotting a 6144 well microplate, each of the 64 dispensations perdispenser tip can be offset from the other dispensations so that eachdispenser tip dispenses to 64 different combinations of X and Ycoordinates, for example, so each tip spots 64 different wells.

In some embodiments, a method of dispensing can comprise: (a) loading adispensing device comprising n fixed dispensers with a first pluralityof fluids from a first source plate, wherein the source plate comprisesm fluids, wherein n is an integer greater than or equal to two, and m isa positive whole number multiple of n; (b) moving a first microplateinto a receiving position with respect to the fixed dispensers; (c)dispensing n fluids from the dispensers onto or into a first set of nlocations on or in the first microplate, (d) moving at least oneadditional microplate into receiving position with respect to thedispensers; (e) dispensing n fluids from the dispensers onto or into afirst set of n locations on or in the at least one additionalmicroplate; (f) loading the n dispensers with a second plurality offluids from a second source plate, wherein the second source platecomprises m fluids; (g) moving the first microplate into a receivingposition with respect to the fixed dispensers; (h) dispensing n fluidsfrom the dispensers onto or into a second set of n locations on or inthe first microplate; (i) moving the at least one additional microplateinto receiving position with respect to the dispensers; and (j)dispensing n fluids from the dispensers onto or into a second set of nlocations on or in the at least one additional microplate. The firstsource plate can be the same as the second source plate, or they can bedifferent source plates.

The method of dispensing can further involve loading from a plurality ofsource plates, for example, four, eight, 16, 32, 64, 96, 384, or more.In some embodiments, the first and second source plates can be the sameand the first plurality of fluids can be a different plurality of fluidsthan the second plurality of fluids. In some embodiments, the firstplurality of fluids can be the same plurality of fluids as the secondplurality of fluids. In some embodiments, the first plurality of fluidscan comprise a first plurality of mixtures, and each mixture cancomprise two or more reagents for a nucleic acid sequence reaction. Themethod can comprise spotting a microplate that comprises, for example,6,144 or more wells.

In some embodiments, a method of dispensing fluids is provided thatcomprises: (a) aspirating a first fluid volume into a dispenser adaptedto dispense fluid volumes of one microliter or less; (b) dispensing adesired amount of the fluid volume, to form a dispensed portion, (c)calculating the volume of the dispensed portion, and (d) calculating anadjusted desired volume that compensates for a difference between thedesired volume and the volume of the dispensed portion. The method canfurther comprise: (e) dispensing an adjusted desired volume of the fluidvolume, to form a second dispensed portion, (f) calculating the volumeof the second dispensed portion, and (g) calculating an adjusted desiredvolume that compensates for a difference between the adjusted desiredvolume and the volume of the dispensed portion. The method can compriserepeating the dispensing and two calculating steps for each dispensationof the dispenser. The method can be used on a piezo-electric dispenser,on an acoustic dispenser, or the like.

The method of dispensing a fluid can comprise calculating the volume byremembering a count of the number of dispensings per aspiration, andlooking up in a table a level of dilution determined by the count. Asfluid can be dispensed from the dispenser, the loss of volume cancomprise an effect on the dispensed amount and the method can improvedispensing accuracy. A computer control unit and a memory can be used totrack the dispensing and determine adjustments to be made ifcompensation is needed for a loss of volume per dispensation. Thedispenser can comprise a plurality of dispensers and the calculating cancomprise calculating a level of dilution of the dispensed volume foreach dispenser of the plurality of dispensers. The dispenser cancomprise a plurality of dispensers and the adjusting can compriseadjusting the dispensed volume of each dispenser of the plurality ofdispensers.

In some embodiments, a method of loading a microplate is provided thatcomprises: translating a filling device comprising a plurality ofdispensers, each dispenser comprising a fluid, along a translation pathtraversing a microplate comprising rows of wells, wherein the wells cancomprise an average minimum dimension equal to a first dimension; anddispensing a band of a respective fluid from each of the dispensersalong a portion of the translation path to load rows of the wells,wherein the bands do not contact one another and the rows include atleast two adjacent loaded rows of wells which can be spaced apart fromone another by a dimension that is about the same as or greater than thefirst dimension. The at least two adjacent loaded rows of wells can beseparated from one another by at least one dead row of wells, that is,at least one row of wells that has not purposefully been loaded, butrather, that may receive some overspray or overshoot of fluids intendedto be dispensed into the loaded wells. In place of a dead row of wells,the method can comprise dispensing to a microplate that includes athickened sidewall between the two adjacent loaded rows, wherein thesidewall can be at least as wide as the average width of each of thewell. The sidewall can be as high as all of the other sidewalls betweenadjacent wells of the microplate.

The method of loading a microplate can comprise the dispensation of, forexample, one or more biological sample. The method can comprise thedispensation of, for example, a biological reagent, an assay, a probe, aprimer, an oligonucleotide, and a combination thereof. The plurality ofthe wells of the microplate can each be preloaded with components for asame kind of assay or for respective different kinds of assays. Eachwell in each row of wells loaded by one of the bands can comprisecomponents for a same kind of assay. In some embodiments, the method cancomprise dispensing a marker fluid in the at least one dead row ofwells, for example, a control liquid, dye, or optical marker. The markerfluid can be used to calibrate fluorescence signals and/or to providefor location identification like a milepost or landmarker.

In some embodiments, loading distribution system 800 can be used totransfer assay components such as oligonucleotides from source plates,for example, 384-well source plates, to microplates 20. Loadingdistribution system 800 can produce a plurality of microplates 20simultaneously in batches. Batches can comprise a plurality of sourceplates, for example, 2, 4, 8, 16, 32, or more source plates. Batches cancomprise a plurality of target microplates, for example, about 5 ormore, about 10 or more, about 100 or more, or about 200 or more,microplates per batch. Loading distribution system 800 can be integratedinto a manufacturing system. The manufacturing system can provide, forexample, work orders, a manufacturing historian, or logger. Themanufacturing system can comprise an enterprise resource planning (ERP)system. Loading distribution system 800 can maintain queues for sourceand target microplates. Loading distribution system 800 can providedifferent temperature and humidity control environments for the sourceand the target microplates. A cache of source and target microplates canbe disposed in appropriate stations of loading distribution system 800.This can allow for the unattended operation of loading distributionsystem 800.

In some embodiments, control software and/or a dispensing device can beutilized that is configurable for a list of variables. Exemplaryvariables can be found herein in the EXAMPLE section. Loadingdistribution system 800 can utilize, for example, a 96-dispenserdispensing device, or a 384-dispenser dispensing device. Loadingdistribution system 800 can utilize, for example, 1, 2, 4, 8, 16, ormore than 16 dispensing devices. Loading distribution system 800 can bedesigned to mitigate a throughput bottleneck at a dispensing device.

In some embodiments, Incoming Quality Control (IQC) requirements formicroplate 20 can be used for a Whole Genome Array (WGA), a Focused GeneSet(s) (FGS) system, or a custom gene-set(s) system. The IQC canrequire, for example, a 100% inspection of a microplate in from about 1second to about 60 seconds, from about 1 second to about 10 seconds, orfrom about 3 seconds to about 6 seconds. The inspection can comprisetests for, for example, an absence or presence of spots, spot metrics,and/or volume and concentration measurements (CPM). The IQC system cancomprise hardware and/or software. In some embodiments, the IQC stationcan comprise a fluorescence detection system using, for example,infrared dye spiking or blue LED excitation of spots. The IQC stationcan be a data logger. The IQC can be a decision maker.

In some embodiments, a dispensing device can be configured to disablerows of dispensers. For example, a 96 dispenser-dispensing device canmimic 12, 24, and 48 dispenser configurations. In some embodiments, theunused dispensers can be disabled, for example, using software. In someembodiments, the unused dispensers can be physically removed from adispense position. A manifold in the dispensing device can bereconfigured to gang disabled tips. A common valve disposed on themanifold can shut-off unused dispensers to prevent them from aspiratingair. The different dispensing devices can be swapped manually orrobotically.

An exemplary loading distribution system can provide many differentcombinations of variables as exemplified in the table below:

Counts Unit Variable number of tips per head 96 number of spotting heads4 number of replicates per tip per source plate well 1 moving timebetween 2 stations 1 sec move time between replicates on microplate 0.5sec tip firing cycle time for each spotting 1 sec number of stations forother functions 4 number of dispenses per tip per source plate 1 numberof high-density microplates per batch 150 number of source plates perbatch 16 number of passes for each microplate 16 volume in tip peraspirate 3 μl volume per dispense 0.03 μl percent of volume dispensedper aspirate 50% number of dispenses per aspirate 50 number of aspiratesper source plate well per tip 3 per batch number of total aspiratecycles per head per batch 12 number of spotting cycles per tip per batch2400 number of spotting cycles per head per batch 2400 number of indexcycles to ramp up and down 14 Total aspirate time per batch 5280 secTotal spotting time per batch 16898 sec Aspirate Serial Actions movewash station in position 5 sec wash tips 45 sec move wash station out 5sec load source plate in aspirate position 5 sec aspirate time 15 secunload source plate from aspirate position 5 sec Aspirate cycle time 80sec Dispense Spotting Station Actions move shuttle in dispense position1 sec position plate for spotting under head 4 sec tip firing time perhigh-density plate per source plate 1 sec reposition plate afterdispense 1 sec Spotting Cycle Times 7 sec Actions load per unload sourceplate @ incubator 40 sec handling time per plate 40 sec Other StationActions move shuttle in dispense position 1 sec unload shuttlehigh-density plate @ hotel 4 sec load high-density plate in shuttle @hotel 4 sec inline QC 4 sec barcode reading and writing of high-densityplate 2 Station process time per pass 5 sec

Loading distribution system 800 can provide the following throughput forspotting with four 96-tip dispense devices.

number of 384-well source plates = 16 16 number of unique assay = 384 ×16 = 6144 number of tips per head = 4 96 number of heads = 4 4 number oftotal tips = 96 × 4 384 number of passes for each high-density plate =6144/4/96 = 64 number of source wells per tip = 6144/384 = 16Microplate Filling

In some embodiments, a filling apparatus 400 can be used to fill atleast some of the plurality of wells 26 of microplate 20 with one ormore components of assay 1000. It should be understood that fillingapparatus 400 can comprise any one of a number of configurations.

In some embodiments, referring to FIGS. 20-22( b), filling apparatus 400comprises one or more assay input ports 402, such as about 96 inputports, disposed in an input layer 404. In some embodiments, assay inputports 402 of input layer 404 can be in fluid communication with aplurality of microfluidic channels 406 disposed in input layer 404, anoutput layer 408, or any other layer of filing apparatus 400. In someembodiments, the plurality of microfluidic channels 406 can be formed inan underside of input layer 404 and a seal member can be placed over theunderside of input layer 404. In some embodiments, the seal member cancomprise a perforation (e.g. hole) positioned over a desired location inmicroplate 20 to permit a discrete fluid communication passage to extendtherethrough. In some embodiments, the plurality of microfluidicchannels 406 can be arranged as a grouping 407 (FIG. 20). In someembodiments, assay input ports 402 can be positioned at a predeterminedpitch (e.g. 9 mm) such that each assay input port 402 can be alignedwith a center of each grouping 407. In some embodiments, the pluralityof microfluidic channels 406 can be in fluid communication with aplurality of staging capillaries 410 formed in output layer 408 (FIGS.21-22( b)).

In some embodiments, input layer 404 and output layer 408 can be bondedor otherwise joined together to form a single unit. This bond can bemade with, among other things, a double-stick tape, a laser weld, anultrasonic weld, or an adhesive. However, it should be appreciated thatthe bonding or otherwise joining of input layer 404 and output layer 408is not required.

During filling, assay 1000 can be put into at least one assay input port402 and can be fluidly channeled toward at least one of the plurality ofmicrofluidic channels 406, first passing a surface tension relief post418 in some embodiments. In some embodiments, surface tension reliefpost 418 can serve, at least in part, to evenly spread assay 1000throughout the plurality of microfluidic channels 406 and/or engage ameniscus of assay 1000 to encourage fluid flow. Assay 1000 can befluidly channeled through the plurality of microfluidic channels 406 andcan collect in the plurality of staging capillaries 410 (FIG. 22( b)).Assay 1000 can then be held in the plurality of staging capillaries 410by capillary or surface tension forces.

In some embodiments, as illustrated in FIGS. 21 and 22( a)-(b),microplate 20 can be attached to filling apparatus 400 so that each ofthe plurality of staging capillaries 410 is generally aligned with eachof the plurality of wells 26. In some embodiments, filling apparatus 400comprises alignment features 411 (FIG. 20) operably sized to engagecorresponding alignment feature 58 on microplate 20 to, at least inpart, facilitate proper alignment of each of the plurality of stagingcapillaries 410 with a corresponding (respective) one of the pluralityof wells 26. In some embodiments, the combined unit of filling apparatus400 and microplate 20 can then be placed in a centrifuge. Thecentrifugal force of the centrifuge can, at least in part, urge assay1000 from the plurality of staging capillaries 410 into each of theplurality of wells 26 of microplate 20. Filling apparatus 400 can thenbe removed from microplate 20. In some embodiments, microplate 20 canthen receive additional reagents and/or be sealed with sealing cover 80,or other sealing feature such as a layer of mineral oil, and then placedinto high-density sequence detection system 10.

In some embodiments, capillary or surface tension forces encourage flowof assay 1000 through staging capillaries 410. In this regard, stagingcapillaries 410 can be of capillary size, for example, stagingcapillaries 410 can be formed with an exit diameter less than about 500micron, and in some embodiments less than about 250 microns. In someembodiments, staging capillaries 410 can be formed, for example, with adraft angle of about 1-5° and can define any thickness sufficient toachieve a predetermined volume. To further encourage the desiredcapillary action in staging capillaries 410, staging capillaries 410 canbe provided with an interior surface that is hydrophilic, i.e.,wettable. For example, the interior surface of staging capillaries 410can be formed of a hydrophilic material and/or treated to exhibithydrophilic characteristics. In some embodiments, the interior surfacecomprises native, bound, or covalently attached charged groups. Forexample, one suitable surface, according to some embodiments, is a glasssurface having an absorbed layer of a polycationic polymer, such aspoly-l-lysine.

Ramps

In some embodiments, as illustrated in FIGS. 22( b) and 23(a)-(b), eachof the plurality of staging capillaries 410 can comprise a ramp feature414 disposed at an entrance thereof to achieve a predetermined capillaryaction. It should be appreciated that ramp feature 414 can be formed onone or more edges of the entrance to each of the plurality of stagingcapillaries 410. In some embodiments, ramp feature 414 can comprise acountersink lip or chamfered rim formed about the entire entrance. Insome embodiments that do not employ the plurality of microfluidicchannels 406, ramp feature 414 can be used to reduce an angle betweenstaging capillary 410 and an upper surface 456 (to be described herein)of output layer 408 to aid in capillary flow and/or exposure time to afluid bead moving thereby.

Nozzles Bottom Features

In some embodiments, with reference to FIGS. 22( b) and 24, output layer408 can comprise a protrusion 450 formed on an outlet 434 of stagingcapillary 410. In some embodiments, protrusion 450 can be shaped tocooperate with a corresponding shape of each of the plurality of wells26. In some embodiments, protrusion 450 can be conically shaped to bereceived within circular rim portion 32 of each of the plurality ofwells 26. In some embodiments, protrusion 450 can be square-shaped to bereceived within square-shaped rim portion 38 of each of the plurality ofwells 26. Protrusion 450, in some embodiments, can define a sufficientlysharp surface such that the capillary force within staging capillary 410can retain assay 1000 and protrusion 450 can inhibit movement of assay1000 to adjacent wells 26. In some embodiments, protrusion 450 of outputlayer 408 can be positioned above microplate 20, flush with firstsurface 22 of microplate 20 (FIG. 22( a)), or disposed within well 26 ofmicroplate 20 (FIG. 22( b)). In some embodiments, protrusion 450 candefine a nozzle feature that comprises a diameter that is less than thediameter of the plurality of wells 26 to aid, at least in part, incapillary retention of assay 1000 within staging capillary 410.

Protrusion 450 can be provided with an exterior surface that ishydrophobic, i.e., one that causes aqueous medium deposited on thesurface to bead. For example, protrusion 450 can be formed of ahydrophobic material and/or treated to exhibit hydrophobiccharacteristics. This can be useful, for example, to prevent spreadingof a drop, formed at tip portion 1840. A variety of known hydrophobicpolymers, such as polystyrene, polypropylene, and/or polyethylene, canbe utilized to obtain desired hydrophobic properties. In addition, or asan alternative, a variety of lubricants or other conventionalhydrophobic films can be applied to tip portion 1840.

Bottom Feature—Spacer

In some embodiments, as illustrated in FIG. 24, one or more spacermembers 452 can be formed along bottom surface 429 of output layer 408to, at least in part, achieve a desired spacing between output layer 408and microplate 20. In some embodiments, spacer member 452 can be formedas an elongated member (FIG. 24), a post (FIG. 107), one or morespaced-apart members, or the like.

Fluidic Patterns

In some embodiments, as illustrated in FIGS. 23( a)-(b) and 25(a)(f),the plurality of microfluidic channels 406 can have any one of aplurality of configurations for carrying assay 1000 to each of theplurality of staging capillaries 410. In some embodiments, each of theplurality of staging capillaries 410 can be in fluid communication withonly one of the plurality of microfluidic channels 406 (FIGS. 23(a)-(b), 25(a)-(d), and 25(f)) in a series-type configuration. In someembodiments, each of the plurality of staging capillaries 410 can be influid communication with two or more of the plurality of microfluidicchannels 406 (FIGS. 25( e)) in a multi-path or parallel-typeconfiguration. In such parallel-type configurations, fluid can flowalong the path of least resistance to fill each of the plurality ofstaging capillaries 410 in the least amount of time. In anyconfiguration, the time required to fill each of the plurality ofstaging capillaries 410 can be reduced by reducing the length of eachmicrofluidic channel 406. In some embodiments, a hybrid of theseries-type and the parallel-type configurations can be used. In someembodiments, as illustrated in FIG. 25( f), each of the plurality ofmicrofluidic channels 406 can be in fluid communication with only oneedge of each of the plurality of staging capillaries 410 to providepass-by and filling action simultaneously.

In some embodiments, each of the plurality of microfluidic channels 406can exert, at least in part, a capillary force to draw fluid (e.g. assay1000) therein to aid in reducing the time required to fill. Thecapillary force of each of the plurality of microfluidic channels 406can be varied, at least in part, by varying at least the dimensionalproperties of the plurality of microfluidic channels 406 according tocapillary principles.

Pressure Nodules

In some embodiments, as illustrated in FIGS. 106-113, filling apparatus400 comprises input layer 404, output layer 408, and an intermediatelayer 494, or any combination thereof for filling one or more componentsof assay 1000 into at least some of the plurality of wells 26 inmicroplate 20.

In some embodiments, intermediate layer 494 can be positioned andaligned between input layer 404 and output layer 408. In someembodiments, input layer 404 comprises assay input ports 402 extendingtherethrough. As illustrated in FIGS. 107 and 108, in some embodiments,each assay input port 402 can extend through input layer 404 andterminate at an extended outlet 496. In some embodiments, extendedoutlet 496 can be sized to extend from input layer 404 such that an end498 of extended outlet 496 is spaced a predetermined distance fromoutput layer 408 (FIG. 108). Extended outlet 496 can extend through acorresponding aperture 500 (FIG. 106) formed through intermediate layer494.

In some embodiments, as illustrated in FIG. 108, extended outlet 496 canbe aligned with surface tension relief post 418 extending upward fromoutput layer 408. In some embodiments, an internal diameter of extendedoutlet 496 can be larger than an outer diameter of surface tensionrelief post 418 to permit surface tension relief post 418 to be at leastpartially received within extended outlet 496. Surface tension reliefpost 418, in some embodiments, can be sufficiently sized to facilitateeven spreading of assay 1000 throughout the plurality of microfluidicchannels 406 and/or engage a meniscus of assay 1000 within assay inputport 402 to encourage flow. In some embodiments, extended outlet 496 andsurface tension relief post 418 can cooperate to facilitate alignmentsof input layer 404, output layer 408, and intermediate layer 494.

In some embodiments, intermediate member 494 comprises microfluidicchannels 406 extending there along (e.g., etched or otherwise formed inone major side thereof) in fluid communication with the plurality ofstaging capillaries 410 in output layer 408. For example, microfluidicchannels 406, extending along a lower surface of intermediate layer 494,can communicate with upper-end openings of staging capillaries 410. Itshould be appreciated that the particular route configuration ofmicrofluidic channels 406 can be any one of a number of configurationsselected by one skilled in the art or one of those described herein. Insome embodiments, intermediate member 494 can be compliant, orresiliently deformable, to permit flexing of intermediate member 494 inresponse to an external force. In some embodiments, intermediate member494 can be made of polymeric materials, such as but not limited torubber or silicone (PDMS).

As illustrated in FIGS. 107-111, in some embodiments, input layer 404comprises one or more nodules 502 extending from a bottom surface 504.In some embodiments, nodules 502 can be patterned along bottom surface504 such that each nodule 502 can engage a top surface 506 of compliantintermediate layer 494. During centrifugation, centripetal force exertedon input layer 404 can cause nodules 502 to engage compliantintermediate layer 494 to at least partially collapse or depress asegment of intermediate layer 494 against output layer 408 to minimizefluid communication between adjacent staging capillaries 410. In someembodiments, as illustrated in FIGS. 109 and 110, nodules 502 can bepatterned such that each nodule 502 is positioned adjacent each of theplurality of staging capillaries 410. For example, nodules 502 can bedisposed so that each nodule aligns, or corresponds, with a respectiveone of staging capillaries 410. In some embodiments, nodules 502 can bepatterned over portions of microfluidic channels 406 to closemicrofluidic channel 406 during centrifugation. In some embodiments, asillustrated in FIG. 111, nodules 502 can be patterned over each of theplurality of staging capillaries 410 to seal each of the plurality ofstaging capillaries 410 during centrifugation. For example, upon beingdepressed by nodules 502 during centrifugation, segments of intermediatelayer 494 can seal the upper end openings of respective, correspondingstaging capillaries 410.

In some embodiments, as illustrated in FIGS. 111 and 112, a sealingfeature 508 can extend from intermediate layer 494 that can be sized tofit into the corresponding staging capillary 410 by nodule 502 actingupon intermediate layer 494. These, and substantially equivalent,embodiments can be used to define a shut-off valve during centrifugationor anytime a force is applied to input layer 404 and/or intermediatelayer 494.

It should be appreciated that the physical size and/or compliancy of oneof more of input layer 404, intermediate layer 494, nodules 502, andsealing features 508 can be tailored to achieve a predetermined sealingengagement upon application of a predetermined amount of force.Additionally, it should be appreciated that nodules 502 and/or sealingfeature 508 can be of any shape conducive to applying a force andsealing an opening, respectively, such as, but not limited to,triangular, square, or conical.

In some embodiments, to load each of the plurality of stagingcapillaries 410, a predetermined amount of assay 1000 can be placed ateach assay input port 402. Capillary force, at least in part, can drawat least a portion of assay 1000 from assay input port 402 intomicrofluidic channels 406 and further fill at least some of theplurality of staging capillaries 410. In some embodiments, once at leastsome of the plurality of staging capillaries 410 are filled, outputlayer 408 and microplate 20 can be placed into a swing-arm centrifuge.In some embodiments, the centripetal force of the swing-arm centrifugecan be sufficient to overcome the surface tension of assay 1000 in eachthe plurality of staging capillaries 410, thereby forcing a meteredvolume of assay 1000 into each of the plurality of wells 26 ofmicroplate 20. In some embodiments, the centripetal force of thecentrifuge can be sufficient to exert a clamping force on at least oneof input layer 404 and intermediate layer 494 to fluidly seal adjacentstaging capillaries 410, either at the entrance thereof or therebetween,to prevent residual assay 1000 left in assay input port 402 or assay1000 from an undesired one of the plurality of wells 26 of microplate 20from overfilling a particular staging capillary. In some embodiments, anexternal force (e.g. mechanical, pneumatic, hydraulic,electro-mechanical, and the like) can be applied to exert a clampingforce on at least one of input layer 404 and intermediate layer 494 tofluidly seal adjacent staging capillaries 410, either at the entrancethereof or therebetween.

In some embodiments, as illustrated in FIG. 113, at least some of inputlayer 404, intermediate layer 494, and output layer 408 can be used inconjunction with a clamp system 511. In some embodiments, clamp system511 comprises a base structure 513 and one or more locking features 515extending therefrom. In some embodiments, base structure 513 comprisesat least one alignment feature 517 operably sized to engage acorresponding alignment feature 58 on microplate 20 to, at least inpart, facilitate proper alignment of each of the plurality of stagingcapillaries 410 relative to each of the plurality of wells 26. In someembodiments, alignment feature 517 can further engage a correspondingalignment feature 519 formed in at least one of input layer 404,intermediate layer 494, and output layer 408. In some embodiments, atleast some of microplate 20, input layer 404, intermediate layer 494,and output layer 408 can be coupled with base structure 513 such thatlocking feature 515 engages input layer 404 to exert a preload onintermediate layer 494 to prevent fluid flow and/or leakage of assay1000 prior to achieving sufficient centrifugal speed in the centrifuge.In some embodiments, a top plate 521 can be used in conjunction withbase structure 513 to ensure equal pressure application across inputlayer 404 by locking feature 515.

Venting

In some embodiments, as illustrated in FIGS. 114-119, filling apparatus400 comprises input layer 404, output layer 408, and a vent layer 523,or any combination thereof for loading assay 1000 into at least some ofthe plurality of wells 26 in microplate 20. In some embodiments, outputlayer 408 comprises microfluidic channels 406 formed in a side thereofand extending there along in fluid communication with the plurality ofstaging capillaries 410 in output layer 408.

In some embodiments, input layer 404 comprises assay input ports 402extending therethrough. As illustrated in FIGS. 115-116, in someembodiments, each assay input port 402 can extend through input layer404 and terminate at extended outlet 496. In some embodiments, extendedoutlet 496 can be sized to extend from input layer 404 such that an end498 of extended outlet 496 is generally flush to a top surface 525 ofvent layer 523 and aligned to a flow aperture 527 extending through ventlayer 523.

In some embodiments, input layer 404 comprises one or more vent features529 (FIGS. 116-119). In some embodiments, vent feature 529 can be sizedto have a capillary force associated therewith that is lower than acapillary force within microfluidic channels 406 and/or each of theplurality of staging capillaries 410 to reduce the likelihood of assay1000 flow through or into vent feature 529. In some embodiments, ventfeature 529 comprises a vent hole 531 extending through input layer 404(FIGS. 114-118) and in communication with atmosphere. In someembodiments, vent hole 531 can be coupled to a chamber or manifold 533(FIGS. 115 and 116) that can couple two or more vent apertures 535formed in vent layer 523 to atmosphere.

In some embodiments, vent feature 529 comprises a pressure bore 537(FIG. 117) associated with one or more of the plurality of stagingcapillaries 410. In some embodiments, pressure bore 537 can be formed ininput layer 404. For example, pressure bore 537 can extend from a lowersurface of input layer 404 toward, but stopping short of, an opposingsurface. In some embodiments, plural pressure bores 537 are disposed inan array corresponding to an array defined by staging capillaries 410.Pressure bores 537, in some embodiments, can be sized to act as an aircapacitor trapping a portion of air therein that can contract or expandduring filling of assay 1000 into filling apparatus 400 and/orcentrifuging assay 1000 into each of the plurality of wells 26,respectively.

Vent feature 529, in some embodiments, can at least partially relievevacuum created when assay 1000 is centrifuged from each of the pluralityof staging capillaries 410 into each of the corresponding plurality ofwells 26 of microplate 20 and permit improved loading. In someembodiments, vent feature 529 can at least partially interrupt fluidflow between adjacent staging capillaries 410 by introducing an air gaptherebetween. In some embodiments, such an air gap can provideconsistent metering of assay 1000 loaded into each of the plurality ofwells 26.

In some embodiments, vent layer 523 can be positioned and alignedbetween input layer 404 and output layer 408. In some embodiments, asillustrated in FIG. 116, flow aperture 527 of vent layer 523 can bealigned with surface tension relief post 418 extending upward fromoutput layer 408. In some embodiments, an internal diameter of flowaperture 527 can be larger than the outer diameter of surface tensionrelief post 418 to permit surface tension relief post 418 to be at leastpartially received within flow aperture 527. Surface tension relief post418, in some embodiments, can be sufficiently sized to facilitate evenspreading of assay 1000 throughout the plurality of microfluidicchannels 406 in output layer 408 and/or engage a meniscus of assay 1000within assay input port 402 and/or flow aperture 527 to encourage flow.In some embodiments, extended outlet 496, flow aperture 527, and surfacetension relief post 418 can cooperate to facilitate alignments of inputlayer 404, output layer 408, and vent layer 523.

As illustrated in FIGS. 116-118, in some embodiments, vent layer 523 canbe aligned with input layer 404 and output layer 408 such that ventapertures 535 are positioned above or between each of the plurality ofstaging capillaries 410. In some embodiments, vent apertures 535 can bea circular bore (FIG. 117) or any other shape, such as oblong (FIG.118), to accommodate for potential misalignment between input layer 404and vent layer 523 and/or potential misalignment between vent layer 523and output layer 408.

In some embodiments, vent layer 523 can be made of any materialconducive to joining with input layer 404 and/or output layer 408. Insome embodiments, vent layer 523 can comprise PDMS, which can aid injoining vent layer 523 to input layer 404 due to the intrinsic tackinessproperties of PDMS. In some embodiments, vent layer 523 can be madeusing a double stick adhesive tape. In such embodiments, the doublestick adhesive tape can be first applied to input layer 404 and thenlaser cut to accurately place vent apertures 535 to simplify assembly ofinput layer 404 and vent layer 523.

In some embodiments, to load each of the plurality of stagingcapillaries 410, a predetermined amount of assay 1000 can be placed ateach assay input port 402. Such placement can be effected, for example,using an automated pipette system (e.g., a Biomek) or hand-operatedsingle- or multi-channel pipette device (e.g., a Pipetman). Capillaryforce, at least in part, can draw at least a portion of assay 1000 fromassay input port 402 into microfluidic channels 406 and further fill atleast some of the plurality of staging capillaries 410. In someembodiments, outlet 434 of each of the plurality of staging capillaries410 permits venting of air within each of the plurality of stagingcapillaries 410 during filling. In some embodiments, once at least someof the plurality of staging capillaries 410 are filled, input layer 404,vent layer 523, output layer 408, and microplate 20 can be placed into aswing-arm centrifuge. In some embodiments, the venting features 529 canreduce vacuum effects on assay 1000 during centrifugation to more easilymeter a volume of assay 1000 into each of the plurality of wells 26 ofmicroplate 20.

Assay Ports on Sides

In some embodiments, as illustrated in FIGS. 120-131, filling apparatus400 can comprise assay input ports 402 positioned within and/or uponoutput layer 408. In some embodiments, as illustrated in FIG. 120, assayinput ports 402 can be positioned at an end 420 of output layer 408. Forexample, such assay input ports can be positioned along a shortdimension of a major surface (e.g., a top surface) of the output layer,adjacent and parallel to an end thereof. In some embodiments, asillustrated in FIG. 121, assay input ports 402 can be positioned at aside 422 of output layer 408. For example, such assay input ports can bepositioned along a long dimension of a major surface (e.g., a topsurface) of the output layer, adjacent and parallel to a side thereof.Still further, in some embodiments, as illustrated in FIG. 122, assayinput ports 402 can be positioned at opposing ends 420 or opposing sides422 (not illustrated) of output layer 408. In some embodiments, assayinput ports 402 can be positioned at opposing ends 420 or opposing sides422 (not illustrated) of output layer 408 with a fluid interrupt 409(e.g. wall or barrier) to fluidly isolate those assay input ports 402 onone end or side from the remaining assay input ports 402 on the otherend or side.

As illustrated in FIG. 123, in some embodiments, assay input ports 402can each comprise a fluid well 424 bound by a plurality of upstandingwalls 426. In some embodiments, fluid well 424 of each assay input port402 can be in fluid communication with one or more correspondingmicrofluidic channels 406 through a throat 430 formed in fluid well 424.For example, such a throat can be formed in a lower region of the fluidwell, so as to fluidly communicate the fluid well with the microfluidicchannels. Throat 430 can comprise a diameter of, for example, 2 mm orless, 1 mm or less, 0.5 mm or less, or 0.25 mm or less. In someembodiments, such as illustrated in FIG. 123, throat 430 comprises areservoir in fluid communication with one or more microfluidic channel406. In some embodiments, surface tension relief post 418 can bedisposed in throat 430 to, at least in part, evenly spread assay 1000throughout the plurality of microfluidic channels 406 and/or engage ameniscus of assay 1000 to encourage fluid flow. Surface tension reliefpost can, according to some embodiments, comprise a hydrophilic surfacein order to further encourage fluid flow into the throat and, thus, themicrochannels.

In some embodiments, as illustrated in at least FIGS. 124-131,microfluidic channels 406 can be in fluid communication with theplurality of staging capillaries 410 extending from microfluidic channel406, through output layer 408, to a bottom surface 429. In someembodiments, bottom surface 429 can be spaced apart from first surface22 of microplate 20 (FIG. 124) or can be in contact with first surface22 of microplate 20. In some embodiments, each of the plurality ofstaging capillaries 410 can be generally aligned with a correspondingone of the plurality of wells 26 of microplate 20. In some embodiments,a protective covering (not shown) can be disposed over microfluidicchannels 406 to provide, at least in part, protection fromcontamination, reduced evaporation, and the like. It should beunderstood that such protective covering can be used with any of thevarious configurations set forth herein.

Referring to FIGS. 125-131, to perform a filling operation, each assayinput port 402 can be at least partially filled with assay 1000 ordifferent assays or fluids (FIG. 125). At least in part throughhydraulic pressure and/or capillary force, assay 1000 can flow fromfluid well 424 of each assay input port 402 through throat 430 into theone or more microfluidic channels 406 (FIG. 126). As assay 1000 flowsacross an end-opening or mouth 432 of each of the plurality of stagingcapillaries 410, capillary action, at least in part, draws a meteredamount of assay 1000 therein (FIG. 127). Assay 1000 can continue to flowdown the one or more microfluidic channels 406 until each of theplurality of staging capillaries 410 can be at least partially filledwith assay 1000 (FIG. 128). In some embodiments, assay 1000 in each ofthe plurality of staging capillaries 410 can be held therein bycapillary or surface tension forces to aid in the equal metering ofassay 1000 to be loaded in each of the plurality of wells 26. In someembodiments, outlet 434 of each of the plurality of staging capillaries410 permits venting of air within each of the plurality of stagingcapillaries 410 during filling.

As illustrated in FIGS. 129 and 130, in some embodiments, fillingapparatus 400 can be stake cut, generally indicated at 435, via device436 along a portion of one or more microfluidic channels 406. In someembodiments, stake-cutting serves to, at least in part, aid in meteringof assay 1000 in each well 26 by isolating the plurality of stagingcapillaries 410 from any excess assay 1000 left in each assay input port402. This arrangement can minimize additional assay 1000 left withineach assay input port 402 from overfilling each of the plurality ofwells 26 during later centrifugation. In some embodiments, stake cuttingcan be completed through mechanical and/or thermal deformation (e.g.heat staking) of output layer 408. It should be appreciated that a Zbigvalve can be used to achieve fluid isolation between the plurality ofstaging capillaries 410 and assay input port 402, such as thosedescribed in commonly-assigned U.S. patent application Ser. No.10/336,274, filed Jan. 3, 2003 and PCT Application No. WO 2004/011147A1.

As illustrated in FIG. 132, in some embodiments, filling apparatus 400can comprise reduced material areas 438 disposed in output layer 408. Insome embodiments, reduced-material areas 438 comprise one or more cutoutportions 440 (e.g. voids, slots, holes, grooves) formed in output layer408 on opposing sides of microfluidic channels 406. The use of reducedmaterial areas 438 can provide, among other things, reduced thermalcapacity in the localized areas, which can increase the rate of heatstaking and/or stake cutting. In some embodiments, the elongated shapeof cutout portion 440 can accommodate any misalignment of the stakingtool relative to output layer 408. In some embodiments, followingstaking, excess assay 1000 in assay input ports 402 and/or the upstreamportion of microfluidic channels 406 relative to stake cut 435 can beremoved, if desired. In some embodiments, this can be accomplished byemploying a wicking member 441, as illustrated in FIG. 131.

In some embodiments, once at least some of the plurality of stagingcapillaries 410 are filled, output layer 408 and microplate 20 can beplaced into a swing-arm centrifuge. In some embodiments, the centripetalforce of the swing-arm centrifuge can be sufficient to overcome thesurface tension of assay 1000 in each the plurality of stagingcapillaries 410, thereby forcing a metered volume of assay 1000 intoeach of the plurality of wells 26 of microplate 20 (FIG. 133).

Referring again to FIGS. 120-122, filling apparatus 400 can beconfigured in any one of a number of configurations as desired. Asdescribed above, as illustrated in FIG. 120, assay input ports 402 canbe positioned at end 420 of output layer 408. When this configuration isused with a microplate comprising 6,144 wells, filling apparatus 400 cancomprise, for example, eight assay input ports 402 that can each be influid communication with eight respective microfluidic channels 406.Each of the eight microfluidic channels 406 can be in fluidcommunication with ninety-six respective staging capillaries 410. Insome embodiments, as illustrated in FIG. 121, assay input ports 402 canbe positioned at side 422 of output layer 408. When this configurationis used with a microplate comprising 6,144 wells, filling apparatus 400can comprise, for example, eight assay input ports 402 that can each bein fluid communication with twelve respective microfluidic channels 406.Each of the twelve microfluidic channels 406 can be in fluidcommunication with sixty-four respective staging capillaries 410. Thisconfiguration can provide shorter channel lengths, which, in somecircumstances, can have more rapid capillary filling times relative tothe configuration of FIG. 120.

In some embodiments, as illustrated in FIG. 122, assay input ports 402can be positioned at opposing ends 420 or opposing sides 422(configuration not illustrated) of output layer 408. When theconfiguration illustrated in FIG. 122 is used with a microplatecomprising 6,144 wells, filling apparatus 400 can comprise, for example,sixteen assay input ports 402 that can each be in fluid communicationwith twelve respective microfluidic channels 406. Each of the twelvemicrofluidic channels 406 can be in fluid communication with thirty-tworespective staging capillaries 410. Likewise, when sixteen assay inputports 402 are positioned along opposing sides 422, sixteen assay inputports 402 can each be in fluid communication with eight respectivemicrofluidic channels 406. Each of the eight microfluidic channels 406can be in fluid communication with forty-eight respective stagingcapillaries 410. These configurations can provide shorter channellengths, which, in some circumstances, can have more rapid capillaryfilling times relative to the configurations of FIGS. 120 and 121.

In some embodiments, the plurality of microfluidic channels 406 can beoriented such that, during centrifugation, they are perpendicular to anaxis of revolution of the centrifuge. In some embodiments, thisorientation can limit the flow of assay 1000 along the plurality ofmicrofluidic channels 406 during centrifugation.

Overfill Solutions

In some embodiments, metering a predetermined amount of assay 1000 intoeach of the plurality of staging capillaries 410 and finally into eachof the plurality of wells 26 can be achieved using a plurality ofoverfill reservoirs disposed in output layer 408. Referring to FIGS.134-139, in some embodiments, filling apparatus 400 comprises fluid well424 in fluid communication with one or more corresponding microfluidicchannels 406 in fluid communication with the plurality of stagingcapillaries 410. In some embodiments, at least one microfluidic channel406 comprises one or more fluid overfill reservoir 442 in fluidcommunication therewith. In some embodiments, the one or more fluidoverfill reservoir 442 can be a bore opened at one end (e.g., a boreextending into output layer 408 from a surface thereof; with the borehaving an open upper-end and a closed bottom end.)

As illustrated in FIGS. 134-139, to perform a filling operation, eachassay input port 402 can be at least partially filled with assay 1000 orother desired fluid (FIG. 134). At least in part through hydraulicpressure and/or capillary force, assay 1000 can flow from fluid well 424of each assay input port 402 into the one or more microfluidic channels406 (FIG. 134). As assay 1000 flows across an upper-end opening or mouth432 of each of the plurality of staging capillaries 410, capillaryaction, at least in part, draws a metered amount of assay 1000 therein(FIG. 135). Assay 1000 can continue to flow down the one or moremicrofluidic channels 406 until each of the plurality of stagingcapillaries 410 can be at least partially filled with assay 1000 (FIG.136). In some embodiments, fluid overfill reservoir 442 can generallyinhibit assay 1000 from flowing into fluid overfill reservoir 442, atleast in part because of the single opening therein generally preventingair within fluid overfill reservoir 442 from exiting. In someembodiments, fluid overfill reservoir can have a diameter equal to thatof staging capillaries 410 and a depth of about 0.05 inch, or less.

In some embodiments, assay 1000 in each of the plurality of stagingcapillaries 410 can be held therein by capillary or surface tensionforces to aid in the equal metering of assay 1000 to be loaded in eachof the plurality of wells 26. In some embodiments, a lower-end openingor open-air outlet 434 of each of the plurality of staging capillaries410 permit venting of air within each of the plurality of stagingcapillaries 410 during filling.

As illustrated in FIGS. 137 and 138 and described above, in someembodiments, filling apparatus 400 can be stake cut, generally indicatedat 435, via device 436 along a portion of one or more microfluidicchannels 406. It should be appreciated that stake-cutting or staking canbe carried out, as previously described.

In some embodiments, once at least some of the plurality of stagingcapillaries 410 are filled, at least output layer 408 and microplate 20can be placed into a swing-arm centrifuge. In some embodiments, thecentripetal force of the centrifuge can be sufficient to overcome thecapillary force and/or surface tension of assay 1000 in each theplurality of staging capillaries 410, thereby forcing a metered volumeof assay 1000 into each of the plurality of wells 26 of microplate 20(FIG. 139). In some embodiments, the centripetal force of the centrifugecan be sufficient to force overfill fluid (e.g. assay 1000 stillremaining in microfluidic channels 406) into overfill reservoir 442,thereby displacing the air within overfill reservoir 442, rather thaninto the plurality of staging capillaries 410. In some embodiments, thisair can serve to isolate one staging capillary 410 from an adjacentstaging capillary 410. In some embodiments, overfill reservoir 442 canact as a reservoir for excess assay 1000. As illustrated in FIG. 140, insome embodiments, overfill reservoir 442 can be disposed within outputlayer 408 and generally aligned with and positioned below at least oneassay input port 402 in output layer 408.

Microfluidic Channel Shapes

As illustrated in FIGS. 141( a)-(g) and 142(a)-(g), in some embodiments,microfluidic channels 406 can have any one or a combination of variousconfigurations. In some embodiments, as illustrated in FIG. 141( a),each microfluidic channel 406 can be in fluid communication with a pairof rows of the plurality of staging capillaries 410 via feeder channels444. In some embodiments, as illustrated in FIGS. 141( b), 142(a), and142(c), microfluidic channel 406 can be in fluid communication with arow of staging capillaries 410 that can be offset to one side ofmicrofluidic channel 406. In some embodiments, as illustrated in FIGS.141( c)-(e) and 142(d)-(f), a cross dimension, e.g., width, ofmicrofluidic channel 406 can vary relative to a diameter of each of theplurality of staging capillaries 410 ranging from larger than thediameter of each staging capillaries 410 to about equal to the diameterof each staging capillaries 410 to less than the diameter of eachstaging capillary (FIGS. 25( e)-(f)). In some embodiments, asillustrated in FIGS. 141( f), 141(g), 142(a), and 142(b), microfluidicchannel 406 can have a generally triangular cross-section that can beeither aligned with or offset from staging capillaries 410. In someembodiments, as illustrated in FIGS. 142( g), microfluidic channel 406can have a single channel portion 446 fluidly coupled to two or morerows of staging capillaries 410. In some embodiments, single channelportion 446 comprises a centrally disposed feature 448 to, in part, aidin fluid splitting between adjacent rows of staging capillaries 410.

In some embodiments, capillary or surface tension forces encourage flowof assay 1000 through microfluidic channels 406. In this regard,microfluidic channels 406 can be of capillary size, for example,microfluidic channels 406 can be formed with a width of less than about500 micron, and in some embodiments less than about 125 microns, lessthan about 100 microns, or less than about 50 microns. In someembodiments, microfluidic channels 406 can be formed, for example, witha depth of less than about 500 micron, and in some embodiments less thanabout 125 microns, less than about 100 microns, or less than about 20microns. To further encourage the desired capillary action inmicrofluidic channels 406, microfluidic channels 406 can be providedwith an interior surface that is hydrophilic, i.e., wettable. Forexample, the interior surface of microfluidic channels 406 can be formedof a hydrophilic material and/or treated to exhibit hydrophiliccharacteristics. In some embodiments, the interior surface comprisesnative, bound, or covalently attached charged groups. For example, onesuitable surface, according to some embodiments, is a glass surfacehaving an absorbed layer of a polycationic polymer, such aspoly-l-lysine.

Floating Inserts

In some embodiments, as illustrated in FIGS. 143-157, filling apparatus400 comprises output layer 408, a floating insert 460, a cover 464, portmember 467, or any combination thereof for loading assay 1000 into atleast some of the plurality of wells 26 in microplate 20.

In some embodiments, output layer 408 comprises one or more recessedregions or depressions 454 formed in an upper surface 456 of outputlayer 408. Each depression 454 can be, in some embodiments, sized and/orshaped to receive floating insert 460 therein. In some embodimentscomprising two or more depressions 454, at least one wall 458 can beused to separate each depression 454 to define grouping 407 of stagingcapillaries 410 of any desired quantity and orientation.

In some embodiments, as illustrated in FIG. 144, floating insert 460 anddepression 454 can together define a capillary gap 468 between a bottomsurface 470 of floating insert 460 and a top surface 472 of depression454. In some embodiments, capillary gap 468 can result from surfacevariations in bottom surface 470 of floating insert 460 and/or topsurface 472 of depression 454 and/or spacing gaps formed therebetween.It should be appreciated that capillary gap 468 can be quite small;therefore, the drawings of the present application may exaggerate thisfeature for ease of printing and understanding. In some embodiments,capillary gap 468 exhibits a capillary force sufficient to draw assay1000 there along and to mouth 432 of each staging capillary 410. In someembodiments, bottom surface 470 of floating insert 460 and/or topsurface 472 of depression 454 can be treated and/or coated to enhancethe hydrophilic properties of capillary gap 468. In some embodiments,capillary gap 468 can be in fluid communication with an aperture 462extend through floating insert 460. Aperture 462 can be centrallylocated relative to floating insert 460 or can be located to one sideand/or corner thereof. In some embodiments, aperture 462 comprises anassay receiving well 463 (FIG. 145-157). In such embodiments, portmember 467 is optional.

As illustrated in FIG. 144, in some embodiments, to reduce capillaryforce between a sidewall 474 of floating insert 460 and wall 458 ofdepression 454, the thickness of floating insert 460 and the depth ofdepression 454 can be minimized to shorten the length of any resultingcapillary channel and, thus, reduce the overall capillary force in thisregion. In some embodiments, as illustrated in FIGS. 145-157, floatinginsert 460 comprises a flanged base portion 490 to reduce the potentialcapillary surface between sidewall 474 of floating insert 460 and wall458 of depression 454. In some embodiments, a hydrophic surface can beemployed between floating insert 460 and wall 458 of depression 454 toreduce capillary force therebetween. In some embodiments, this hydrophicsurface can result from native material characteristics, treatments,coatings, and the like.

In some embodiments, as illustrated in FIGS. 147-152, floating insert460 can be shaped to, at least in part, achieve any particular capillaryand/or flow characteristics. In some embodiments, as illustrated inFIGS. 147-149, floating insert 460 can comprise a plurality of flowfeatures 478 to, at least in part, extend the capillary surface tofacilitate capillary flow. In some embodiments, for example, each of theplurality of flow features 478 comprises a post member 480 (FIG. 147)extending orthogonally from bottom surface 470 of floating insert 460.In some embodiments, post member 480 comprises a radiused root portion482 to facilitate capillary flow, if desired. In some embodiments, postmember 480 can be offset within the corresponding staging capillary 410and can, if desired, contact a sidewall of staging capillary 410. Insome embodiments, each of the plurality of flow features 478 comprises atapered member 484 (FIGS. 148-152) extending from bottom surface 470 offloating insert 460. In some embodiments, each of the plurality ofstaging capillaries 410 comprises a corresponding mating entrancefeature 486 (FIGS. 148, 150, and 151) to closely conform to each flowfeature 478 to define a transition capillary gap 488. Tapered member 484can be conically shaped (FIGS. 148-149) to closely conform to thecomplementarily-shaped mating entrance feature 486 in staging capillary410. It should be appreciated that in some embodiments, the plurality offlow features 478 can further serve to individually plug or seal eachcorresponding capillary 410 during centrifugation (FIG. 152).

In some embodiments, floating insert 460 can comprise any materialconducive to encourage capillary action along capillary gap 468, such asbut not limited to plastic, glass, elastomer, and the like. In someembodiments, floating insert 460 can be made of at least two materials,such that an upper portion can be made of a first material and a lowerportion can be made of a second material. In some embodiments, thesecond material can provide a desired compliancy, hydrophilicity, or anyother desire property for improved fluid flow and/or sealing of stagingcapillaries 410. In some embodiments, the tapered members can include aseal-facilitating film, coating, or gasket thereon.

In some embodiments, as seen in FIG. 144, cover 464 can be used, atleast in part, to retain floating insert 460 within each depression 454,if desired. In some embodiments, cover 464 comprises an aperture 466generally aligned with an aperture 462 of floating insert 460. In someembodiments, cover 464 comprises a pressure sensitive adhesive to, atleast in part, retain floating insert 460 within depression 454.

As illustrated in FIGS. 143 and 144, in some embodiments, port member467 comprises assay input port 402. In some embodiments, port member 467can comprise a material comprising sufficient weight such that duringcentrifugation, the centripetal force of port member 467 exerted uponfloating insert 460 and output layer 408 can aid in closing offcross-communication of fluid between adjacent staging capillaries 410,as the upper-end openings of staging capillaries 410 can be covered andsealed by the lower surface of floating insert 460. In some embodiments,port member 467 can be sized such that its footprint (e.g. the surfacearea of a bottom surface 476 of port member 467) can be smaller than theopening of depression 454 to aid in the exertion of centripetal force onfloating insert 460 during centrifuge.

In some embodiments, as illustrated in FIG. 153-155, to load each of theplurality of staging capillaries 410, a predetermined amount of assay1000 can be placed at each assay input port 402 when used with portmember 467 or receiving well 463. Capillary gap 468 can be sized toprovide sufficient capillary force to draw at least a portion of assay1000 from assay input port 402 or receiving well 463 into capillary gap468. The capillary force of capillary gap 468 can be, at least in part,due to the non-rigid connection between floating insert 460 and outputlayer 408. As illustrated in FIG. 154, as assay 1000 is drawn into andspreads about capillary gap 468, each of the plurality of stagingcapillaries 410 in fluid communication with capillary gap 468 can beginto fill, at least in part, by capillary force as described herein.

In some embodiments, once at least some of the plurality of stagingcapillaries 410 are filled, at least output layer 408 and microplate 20can be placed into a centrifuge. For example, the pieces can be clampedor otherwise held together, and then placed in a bucket centrifuge as aunit. In some embodiments, the centripetal force of the centrifuge canbe sufficient to overcome the capillary force and/or surface tension ofassay 1000 in each the plurality of staging capillaries 410, therebyforcing a metered volume of assay 1000 into each of the plurality ofwells 26 of microplate 20. In some embodiments, the centripetal force ofthe centrifuge can also cause floating insert 460 to be forced and,thus, pressed against top surface 472 of depression 454. In someembodiments, where port member 467 is installed (FIGS. 143 and 144) orany additional weight member 492 (FIGS. 156 and 157), this additionalweight can further apply a force upon floating insert 460 to forcefloating insert 460 against top surface 472 of depression 454. Thisforce on floating insert 460 against top surface 472 of depression 454can help to fluidly isolate each staging capillaries 410 from adjacentstaging capillaries 410 for improved metering.

It should be appreciated that any component of filling apparatus 400,such as input layer 404, output layer 408, floating insert 460, cover464, port member 467, intermediate layer 494, vent layer 523, etc., cancomprise a plate, tile, disk, chip, block, wafer, laminate, and anycombinations thereof, and the like.

Surface Wipe

As illustrated, for example, in FIGS. 158-166, in some embodiments,filling apparatus 400 does include the plurality of microfluidicchannels 406. In some embodiments, for example, filling apparatus 400comprises output layer 408 and a surface wipe assembly 1800 for loadingassay 1000 into at least some of the plurality of wells 26 in microplate20. In some embodiments, surface wipe assembly 1800 comprises one ormore of a base support 1810, a drive assembly 1812, a funnel assembly1814, or any combination thereof.

In some embodiments, such as illustrated in FIG. 158, base support 1810can be a generally planar support member operable to support microplate20 and output layer 408 thereon. In some embodiments, base support 1810comprises an alignment feature 1818 that can engage correspondingalignment feature 58 (refer to previous figures) of microplate 20 and/oralignment feature 519 of output layer 408 to maintain microplate 20 andoutput layer 408 in a predetermined alignment relative to each otherand/or funnel assembly 1814.

In some embodiments, drive assembly 1812 comprises a drive motor 1816; aguide member 1820, coupled to or formed in base support 1810; a trackingmember 1822, coupled to or formed in funnel assembly 1814; and controlsystem 1010. In some embodiments, guide member 1820 and tracking member1822 are sized and/or shaped to slidingly engage with each other toprovide guiding support for funnel assembly 1814 as it moves relative tobase support 1810. In some embodiments, drive motor 1816 can be operablycoupled to tracking member 1822 or base support 1810 to move trackingmember 1822 relative to guide member 1820 via known drive transmissioninterfaces, such as mechanical drives, pneumatic drives, hydraulicdrives, electromechanical drives, and the like. In some embodiments,drive motor 1816 can be controlled in response to control signals fromcontrol system 1010 or a separate control system. In some embodiments,drive motor 1816 can be operably controlled in response to a switchdevice controlled by a user.

In some embodiments, funnel assembly 1814 comprises a spanning portion1824 generally extending above output layer 408. In some embodiments,spanning portion 1824 can be supported on opposing ends by trackingmember 1822 of drive assembly 1812 and a foot member 1826. Trackingmember 1822 and foot member 1826 can each be coupled to spanning portion1824 via conventional fasteners in some embodiments. Foot member 1826can be generally arcuately shaped so as to reduce the contact areabetween foot member 1826 and base support 1810. In some embodiments,foot member 1826 can be made of a reduced friction material, such asDelrin®.

In some embodiments, spanning portion 1824 of funnel assembly 1814comprises a slot 1828 formed vertically therethrough that can be sizedand/or shaped to receive a funnel member 1830 therein. As illustrated inFIGS. 158-166, funnel member 1830 can comprise one or more assaychambers 1832 for receiving one or more different assays therein. Itshould be appreciated that drive assembly 1812 and funnel assembly 1814can be configured to track in a direction perpendicular to thatillustrated in the accompanying figures to provide an increased numberof assay chambers 1832 and reduced track distances. In some embodiments,such as illustrated in FIG. 159, funnel member 1830 can comprise aflange portion 1834 extending about a top portion thereof. Flangeportion 1834 of funnel member 1830 can be sized and/or shaped to restupon a corresponding flange portion 1836 of slot 1828 of spanningportion 1824 to support funnel member 1830. However, it should beappreciated that funnel member 1830 can comprise any outer profilecomplementary to slot 1828.

Assay chambers 1832, in some embodiments, can be shaped to provide apredetermined assay capacity for filling all of a predetermined numberand/or grouping of the plurality of staging capillaries 410 in outputlayer 408. In some embodiments, assay chamber 1832 comprises convergingsidewalls 1838 that terminate at a tip portion 1840.

In some embodiments, such as illustrated in FIG. 160-162, to load eachof the plurality of staging capillaries 410, a predetermined amount ofassay 1000 can be placed in each assay chamber 1832. In someembodiments, each assay chamber 1832 comprises a different assay. Assay1000 is drawn down along sidewalls 1838 to tip portion 1840 to form afluid bead 1842 extending from tip portion 1840 that can be in contactwith upper surface 456 of output layer 408. In some embodiments, fluidbead 1842 can be bound by a lip or wiper member 1844 extendingdownwardly from tip portion 1840 of funnel member 1830. In someembodiments, wiper member 1844 can, at least in part, wipe and/or removeexcess assay 1000 on upper surface 456 of output layer 408 as funnelmember 1830 moves thereabout. In some embodiments, drive assembly 1812can be actuated to advance funnel assembly 1814 across output layer 408at a predetermined rate, as illustrated in FIG. 161. However, it shouldbe appreciated that funnel assembly 1814 can be advanced manually acrossoutput layer 408. As funnel assembly 1814 is advanced across outputlayer 408, in some embodiments, fluid bead 1842 can contact theupper-end opening or entrance of each of the plurality of stagingcapillaries 410 and begin to fill, at least in part, by capillary forceas described herein.

In some embodiments, such as illustrated in FIGS. 158 and 162, as funnelassembly 1814 continues past the last of the plurality of stagingcapillaries 410, some assay 1000 can be forced off upper surface 456 ofoutput layer 408 at an edge 1846 into at least one overflow channel1848. In some embodiments, once at least some of the plurality ofstaging capillaries 410 are filled, at least output layer 408 andmicroplate 20 can be placed into a centrifuge. In some embodiments, thecentripetal force of the centrifuge can be sufficient to overcome thecapillary force and/or surface tension of assay 1000 in each theplurality of staging capillaries 410, thereby forcing a metered volumeof assay 1000 into each of the plurality of wells 26 of microplate 20.

In some embodiments, such as illustrated in FIG. 158, the excess assay1000 in overflow channel 1848 can be contained using one or morereservoir pockets 1850. In some embodiments, reservoir pocket 1850 canbe in fluid communication with at least one overflow channel 1848. Insome embodiments, reservoir pocket 1850 can be deeper than overflowchannel 1848 to encourage flow of assay 1000 to reservoir pocket 1850.During centrifugation, centripetal force can further encourage assay1000 to flow to reservoir pocket 1850, thereby reducing the likelihoodof any contamination or cross-feed between adjacent staging capillaries410. In some embodiments, an extended wall member 1852 can be positionedabout reservoir pocket 1850 to further contain assay 1000.

In some embodiments, such as illustrated in FIGS. 163 and 164, theexcess assay 1000 in overflow channel 1848 can be contained using areservoir trough 1854. In some embodiments, an absorbent member 1856 canbe disposed in reservoir trough 1854 to absorb excess assay 1000therein. In some embodiments, absorbent member 1856 can be a hydrophilicfiber membrane. As illustrated in FIG. 164, reservoir trough 1854 can besloped toward absorbent member 1856 to facilitate absorption of excessassay 1000. In some embodiments, absorbent member 1856 can be removableto permit removal and relocating of the excess assay 1000 prior tocentrifugation.

In some embodiments, such as illustrated in FIGS. 165 and 166, funnelmember 1830 can comprise two or more discrete assay chambers 1832 fordelivering one or more different assays. In such embodiments, forexample, output layer 408 can comprise one or more central overflowchannels 1858 extending along upper surface 456 of output layer 408 toreceive at least some overflow assay 1000. In some embodiments, centraloverflow channels 1858 are each disposed between each separate groupingof staging capillaries 410 served by each discrete assay chamber 1832.In some embodiments, as illustrated in FIG. 166, central overflowchannel 1858 can be sloped down to at least one of overflow channel 1848(FIG. 158), reservoir pocket 1850 (FIG. 158), reservoir trough 1854(FIG. 163), or absorbent member 1856 (FIG. 166). As illustrated in FIG.165, in some embodiments, absorbent member 1856 can be sized and/orshaped to fit with an enlarged reservoir pocket 1850.

Funnel Member

As illustrated in FIGS. 167-180, in some embodiments, funnel member 1830of funnel assembly 1814 can be any one of a number of configurationssufficient to maintain fluid bead 1842 in contact with upper surface 456of output layer 408. In some embodiments, a predetermined shape of fluidbead 1842 and/or a predetermined flowrate of assay 1000 through tipportion 1840 can be achieved through the particular configuration offunnel member 1830.

As illustrated in FIG. 167-169, in some embodiments, funnel member 1830comprises one or more assay chambers 1832 in fluid communication withtip portion 1840. As described above, in embodiments comprising two ormore assay chambers 1832 (FIG. 168), multiple assays can be used suchthat a different assay can be disposed in each assay chamber 1832. Itshould be understood that any number of assay chambers 1832 can be used(e.g., 2, 4, 6, 8, 10, 12, 16, 20, 32, 64, or more).

In some embodiments, tip portion 1840 can be configured to define acapillary force and/or surface tension sufficient to prevent assay 1000from exiting assay chamber 1832 prior to fluid bead 1842 engaging uppersurface 456 and to permit assay 1000 to be pulled into each of theplurality of staging capillaries 410 during filling of the stagingcapillaries. As illustrated in FIG. 170, tip portion 1840 comprises arestricted orifice 1860 that is sized to increase surface tension toretain assay 1000 with assay chamber 1832. In some embodiments, tipportion 1840 can be spaced apart from an underside surface 1862 to, atleast in part, inhibit assay 1000 from collecting between funnel member1830 and output layer 408. In some embodiments, as illustrated in FIG.171, restricted orifice 1860 can be used with wiper member 1844 toincrease surface tension to retain assay 1000 and to wipe and/or removeexcess assay 1000 on upper surface 456 of output layer 408. In someembodiments, such as illustrated in FIG. 172, tip portion 1840 cancomprise a planar cavity 1864 disposed in fluid communication withrestricted orifice 1860. In some embodiments, planar cavity 1864 canencourage the formation of wider and/or shallower fluid bead 1842relative to similar configurations not employing planar cavity 1864. Insome configurations, the wider and/or shallower fluid bead 1842 can, atleast in part, prolong the time fluid bead 1842 is in contact with eachof the plurality of staging capillaries 410.

As illustrated in FIG. 173, in some embodiments, funnel member 1830 cancomprise wiper 1844 spaced apart from tip portion 1840 to wipe and/orremove excess assay 1000 on upper surface 456 of output layer 408. Insome embodiments, wiper 1844 can extend a distance from undersidesurface 1862 of funnel member 1830 equal to about a distance fromunderside surface 1862 to a distal end of tip portion 1840. Asillustrated in FIGS. 174-176, each tip portion 1840 associated with eachassay chamber 1832 can be offset relative to adjacent tip portions 1840.In some embodiments, this offset relationship between adjacent tipportions 1840 can permit the plurality of staging capillaries 410 to beclosely spaced with reduced likelihood for crosstalk between adjacentfluid beads 1842.

Still referring to FIGS. 174-176, in some embodiments, restrictedorifice 1860 comprises an elongated slot 1866 (FIG. 174) generallyextending from one edge of tip portion 1840 to the opposing edge todefine an elongated fluid bead 1842. However, in some embodiments,restricted orifice 1860 comprises one or more apertures 1868. In someembodiments, the reduced cross-sectional area of apertures 1868 relativeto that of elongated slot 1866 can serve to withstand a fluid headpressure exerted by assay 1000 in assay chamber 1832 that wouldotherwise overcome the surface tension of fluid bead 1842 exitingelongated slot 1866 and possibly lead to premature discharge of assay1000. In some embodiments, the restricted orifice 1860 can be collinearas well as offset as illustrated in (FIG. 174).

In some embodiments, such as illustrated in FIGS. 177-179, funnel member1830 can comprise an internal siphon passage 1870 to, at least in part,control the flowrate of assay 1000 from restricted orifice 1860. In someembodiments, funnel member 1830 comprises a main chamber 1872 fluidlycoupled to a delivery chamber 1874 via siphon passage 1870. In someembodiments, siphon passage 1870 can be positioned along a bottom ofmain chamber 1872. Siphon passage 1870 can comprise an upturned section1876 that can require assay 1000 in main chamber 1872 to flow, at leastin part, against the force of gravity. In some embodiments, main chamber1872 and delivery chamber 1874 can be fluidly coupled at the top thereofby a top chamber 1878. When main chamber 1872 is filled at leastpartially above top chamber 1878, the excess assay 1000 can flow acrosstop chamber 1878 into delivery chamber 1874. During filling, as thelevel of assay 1000 drops below the bottom surface of top chamber 1878and assay 1000 flows from restricted orifice 1860, assay 1000 withindelivery chamber 1874 can be replaced through the siphoning action ofsiphon passage 1870 at the bottom of main chamber 1872. This arrangementcan reduce the fluid head pressure exerted at restricted orifice 1860.Accordingly, the fluid head pressure exerted at restricted orifice 1860can be generally to about the fluid head pressure of assay 1000contained in delivery chamber 1874.

In some embodiments, as illustrated in FIGS. 179 and 180, funnel member1830 can be formed with a two- or more-piece construction. Asillustrated in FIG. 179, funnel member 1830 can comprise a first section1880 and a second section 1882. First section 1880 can comprise one ormore desired features. For example, as illustrated in FIG. 179, upturnedsection 1876 of FIG. 178 can be formed in first section 1880. Firstsection 1880 and second section 1882 can then be joined or otherwisemated along a generally vertical joining line 1884 (FIG. 178) to formfunnel member 1830. In some embodiments, first section 1880 and secondsection 1882 can be joined or otherwise mated along a generallyhorizontal joining line 1886 (FIG. 180). In some embodiments, firstsection 1880 and second section 1882 can be made from differentmaterials to achieve a predetermined performance. In some embodiments,second section 1882 can be made of an elastomer to provide enhanceflexibility to accommodate for variations in output layer 408 andenhanced wiping performance of wiper member 1844.

Surface Treatment

In some embodiments, portions of filling apparatus 400 that are intendedto contact assay 1000, such as assay input ports 402, microfluidicchannels 406, the plurality of staging capillaries 410, and the like,can be hydrophilic. Likewise, in some embodiments, surfaces not intendedto contact assay 1000 can be hydrophobic.

In some embodiments, filling apparatus 400 comprises a treatment toincrease surface energy thereof to improve flow and/or capillary actionof any surface of filling apparatus 400 exposed to assay 1000, such asassay input ports 402, microfluidic channels 406, staging capillaries410, microfluidic channels 406, depression 454, upper surface 456, etc.In some embodiments, surface energy can be improved, for example, whenusing a polymer material in the manufacture of filling apparatus 400,through surface modification of the polymer material via Michaeladdition of acrylamide or PEO-acrylate onto laminated surface; surfacegrafting of acrylamide or PEO-acrylate via atom transfer radicalpolymerization (ARTP); surface grafting of acrylamide via Ce(IV)mediated free radical polymerization; surface initiated living radicalpolymerization on chloromethylated surface; coating of negativelycharged polyelectrolytes; plasma CVD of acrylic acid, acrylamide, andother hydrophilic monomers; or surface adsorption of an ionic ornon-ionic surfactant. In some embodiments, surfactants, such as thoseset forth in Tables 2 and 3, can be used.

TABLE 2 Surfactants for Coating Hydrophile-Lipophile No. Name MW Balance(HLB) 1 Tetronic 901 4700 3 2 Tetronic 1107 1500 24 3 Tetronic 1301 68002 4 Poly(styrene-b-ethylene oxide) Mn: 3600-67000 5Poly(stryrene-b-sodium acrylate) Mn: 1800-42500 6 Triton X-100 13.5 7Triton X-100 reduced 8 Tween 20 1228 16.7 9 Tween 85 1839 11 10 Span 831109.56 3.7 11 Span 80 428.62 4.3 12 Span 40 402.58 6.7 Tetronic:

Triton X-100:

Triton X-100 reduced:

Span 80:

Spam 83:

Spam 20:

Tween: Poly(oxyethylene) sorbitan monolauate

TABLE 3 Surfactants for Wetting Polypropylene Acids: Dodecyl sulfate, Nasalt CH₂(CH₂)₁₁OSO₃ ⁻Na⁺ Octadecyl sulfate, Na salt CH₃(CH₂)₁₇OSO₃ ⁻Na⁺Quaternary ammonium compounds: Cetyltrimethylammonium bromideCH₃(CH₂)₁₅N⁺(CH₃)₃Br⁻ Octadecyltrimethyl ammonium bromideCH₃(CH₂)₁₇N⁺(CH₃)₃Br⁻ Ethers: Brij-52 CH₃(CH₂)₁₅(OCH₂CH₂)₂OH Brij 56CH₃(CH₂)₁₅(OCH₂CH₂)₁₀OH Brij 58 CH₃(CH₂)₁₅(OCH₂CH₂)₂₀OH Brij 72CH₃(CH₂)₁₇(OCH₂CH₂)₂OH Brij 76 CH₃(CH₂)₁₇(OCH₂CH₂)₁₀OH Brij 78CH₃(CH₂)₁₇(OCH₂CH₂)₂₀OH Esters: Poly(ethylene glycol) monolaurateCH₃(CH₂)₁₀CO(OCH₂CH₂)_(4.5)OH Poly(ethylene glycol) distearateCH₃(CH₂)₁₆—CO—(OCH₂)₉—O—CO—(CH₂)₁₆CH₃ Poly(ethylene glycol)dioleateCH₃(CH₂)₇CH═CH(CH₂)₇—CO—(OCH₂)₉—O—CO—(CH₂)₇CH═CH(CH₂)₇CH₃

In some embodiments, filling apparatus 400 can comprise polyolefins;poly(cyclic olefins); polyethylene terephthalate;poly(alkyl(meth)acrylates); polystyrene; poly(dimethyl siloxane);polycarbonate; structural polymers, for example, poly(ether sulfone),poly(ether ketone), poly(ether ether ketone), and liquid crystallinepolymers; polyacetal; polyamides; polyimides; poly(phenylene sulfide);polysulfones; poly(vinyl chloride); poly(vinyl fluoride);poly(vinylidene fluoride); copolymers thereof; and mixtures thereof.

In some embodiments, a co-agent can be employed to enhance thehydrophilicity and/or improve the shelf life of filling apparatus 400.Co-agents can be, for example, a water-soluble or slightly water-solublehomopolymer or copolymers prepared by monomers comprising, for example,(meth)acrylamide; N-methyl(methyl)acrylamide,N,N-dimethyl(methyl)acrylamide, N-ethyl(meth)acrylamide,N-n-propyl(meth)acrylamide, N-iso-propyl(meth)acrylamide,N-ethyl-N-methyl(meth)acrylamide, N,N-diethyl(meth)acrylamide,N-hydroxymethyl(meth)acrylamide, N-(3-hydroxypropyl)(meth)acrylamide,N-vinylformamide, N-vinylacetamide, N-methyl-N-vinylacetamide, vinylacetate that can be hydrolyzed to give vinylalcohol afterpolymerization, 2-hydroxyethyl (meth)acrylate,3-hydroxypropyl(meth)acrylate, N-vinypyrrolidone, poly(ethylene oxide)(meth)acrylate, N-(meth)acryloxysuccinimide, N-(meth)acryloylmorpholine,N-2,2,2-trifluoroethyl(meth)acrylamide, N-acetyl(meth)acrylamide,N-amido(meth)acrylamide, N-acetamido(meth)acrylamide,N-tris(hydroxymethyl)methyl(meth)acrylamide,N-(methyl)acryloyltris(hydroxymethyl)methylamine, (methyl)acryloylurea,vinyloxazolidone, vinylmethyloxazolidone, and combinations thereof. Insome embodiments, the co-agent can be poly(acrylicacid-co-N,N-dimethylacrylamide) or poly(N,N-dimethylacrylamide-co-styrene sulfonic acid).

Microplate Sealing Cover

In some embodiments, such as illustrated in FIGS. 26 and 27, sealingcover 80 can be generally disposed across microplate 20 to seal assay1000 within each of the plurality of wells 26 of microplate 20 along asealing interface 92 (see FIGS. 4, 5, 26, and 27). That is, sealingcover 80 can seal (isoloate) each of the plurality of wells 26 and itscontents (i.e. assay 1000) from adjacent wells 26, thus maintainingsample integrity between each of the plurality of wells 26 and reducingthe likelihood of cross contamination between wells. In someembodiments, sealing cover 80 can be positioned within an optionaldepression 94 (FIG. 30) formed in main body 28 of microplate 20 topromote proper positioning of sealing cover 80 relative to the pluralityof wells 26.

In some embodiments, sealing cover 80 can be made of any materialconducive to the particular processing to be done. In some embodiments,sealing cover 80 can comprise a durable, generally optically transparentmaterial, such as an optically clear film exhibiting abrasion resistanceand low fluorescence when exposed to an excitation light. In someembodiments, sealing cover 80 can comprise glass, silicon, quartz,nylon, polystyrene, polyethylene, polycarbonate, copolymer cyclicolefin, polycyclic olefin, cellulose acetate, polypropylene,polytetrafluoroethylene, metal, and combinations thereof.

In some embodiments, sealing cover 80 comprises an optical element, suchas a lens, lenslet, and/or a holographic feature. In some embodiments,sealing cover 80 comprises features or textures operable to interactwith (e.g., by interlocking engagement) circular rim portion 32 orsquare-shaped rim portion 38 of the plurality of wells 26. In someembodiments, sealing cover 80 can provide resistance to distortion,cracking, and/or stretching during installation. In some embodiments,sealing cover 80 can comprise water impermeable-moisture vaportransmission values below 0.5 (cc-mm)/(m2-24 hr-atm). In someembodiments, sealing cover 80 can maintain its physical properties in atemperature range of 4° C. to 99° C. and can be generally free ofinclusions (e.g. light blocking specks) greater than 50 μm, scratches,and/or striations. In some embodiments, sealing cover 80 can comprise aliquid such as, for example, oil (e.g., mineral oil).

In some embodiments, such sealing material can comprise one or morecompliant coatings and/or one or more adhesives, such as pressuresensitive adhesive (PSA) or hot melt adhesive. In some embodiments, apressure sensitive adhesive can be readily applied at low temperatures.In some embodiments, the pressure sensitive adhesive can be softened tofacilitate the spreading thereof during installation of sealing cover80. In some embodiments, such sealing maintains sample integrity betweeneach of plurality of wells 26 and prevents wells cross-contamination ofcontents between wells 26. In some embodiments, adhesive 88 exhibits lowfluorescence.

In some embodiments, the sealing material can provide sufficientadhesion between sealing cover 80 and microplate 20 to withstand about2.0 lbf per inch or at least about 0.9 lbf per inch at 95° C. In someembodiments, the sealing material can provide sufficient adhesion atroom temperature to contain assay 1000 within each of the plurality ofwells 26. This adhesion can inhibit sample vapor from escaping each ofthe plurality of wells 26 by either direct evaporation or permeation ofwater and/or assay 1000 through sealing cover 80. In some embodiments,the sealing material maintains adhesion between sealing cover 80 andmicroplate 20 in cold storage at 2° C. to 8° C. range (non-freezingconditions) for 48 hours.

In some embodiments, in order to improve sealing of the plurality ofwells 26 of microplate 20, various treatments to microplate 20 can beused to enhance the coupling of sealing cover 80 to microplate 20. Insome embodiments, microplate 20 can be made of a hydrophobic material orcan be treated with a hydrophobic coating, such as, but not limited to,a fluorocarbon, PTFE, or the like. The hydrophobic material or coatingcan reduce the number of water molecules that compete with the sealingmaterial on sealing cover 80. As discussed above, grooves 52, 54 can beused to provide seal adhesion support on the outer edges of sealingcover 80. In these embodiments, for example, a pressure chamber gasketcan be sealed against grooves 52, 54 for improved sealing.

Turning now to FIG. 28, in some embodiments, sealing cover 80 cancomprise multiple layers, such as a friction reduction film 82, a basestock 84, a compliant layer 86, a pressure sensitive adhesive 88, and/ora release liner 90. In some embodiments, friction reduction film 82 canbe Teflon or a similar friction reduction material that can be peeledoff and removed after sealing cover 80 is applied to microplate 20 andbefore microplate 20 is placed in high-density sequence detection system10. In some embodiments, base stock 84 can be a scuff resistant andwater impermeable layer with low to no fluorescence. While in someembodiments, compliant layer 86 can be a soft silicone elastomer orother material known in the art that is deformable to allow pressuresensitive adhesive 88 to conform to irregular surfaces of microplate 20,increase bond area, and resist delamination of sealing cover 80. In someembodiments, pressure sensitive adhesive 88 and compliant layer 86 canbe a single layer, if the pressure sensitive adhesive exhibit sufficientcompliancy. Release liner 90 is removed prior to coupling pressuresensitive adhesive 88 to microplate 20.

Compatibility of Cover and Assay

In some embodiments, adhesive 88 can selected so as to be compatiblewith assay 1000. For example, in some embodiments adhesive 88 is free ofnucleases, DNA, RNA and other assay components, as discussed below. Insome embodiments, sealing cover 80 comprises one or more materials thatare selected so as to be compatible with detection probes in assay 1000.In some embodiments, adhesive layer 88 is selected for compatibilitywith detection probes.

Methods of matching a detection probe with a compatible sealing cover 80include, in some embodiments, varying compositions of sealing cover 80by different weight percents of components such as polymers,crosslinkers, adhesives, resins and the like. These sealing covers 80can then be tested as a function of their corresponding fluorescentintensity level for different dyes. In such embodiments, comparison canbe analyzed at room temperature as well as at elevated temperaturestypically employed with PCR. Comparisons can be analyzed over a periodof time and in some embodiments, the time period can be, for example, upto 24 hours. Data can be collected for each of the varying compositionsof sealing cover 80 and plotted such that fluorescence intensity of thedye is on the X-axis and time is on the Y-axis. Some embodiments of thepresent teachings include a method of testing compatibility of thedetection probe comprising an oligonucleotide and a fluorophore to acomposition of a sealing cover. In such embodiments, the method includesdepositing a quantity of the fluorophore into a plurality of containers,providing a plurality of sealing covers that have different compositionsand sealing the containers with the sealing covers. Methods also includeexciting the fluorophore in each of the containers and then measuring anemission intensity from the fluorophore in each of the containers. Insuch embodiments, the method can also include an evaluation of theemission intensity from the fluorophore of each of the containers andthen a determination of which sealing cover composition is compatiblewith the fluorophore. In some embodiments, the method includes holding atemperature of the containers constant. The method can include measuringthe emission intensity from the fluorophore in each container over aperiod of time, for example, as long as about 24 hours. In someembodiments, the method includes heating the containers to a temperatureabove about 20° C., optionally to a temperature from about 55° C. toabout 100° C. In some embodiments, the method includes cycling thetemperature of the plurality of containers. The temperature of thecontainers can be cycled according to a typical PCR temperature profile.Table 4 shows exemplary data that can be generated for such acomparison. In this example, a dye is evaluated by comparing it atnon-heated and heated temperatures to a cyclic olefin copolymer (COC)and glue material with varying percentages of a crosslinker.

TABLE 4 Percentage of Flourescence Signal Loss Percentage ofFluorescence Signal Loss Post Incubation with Dye (20 hrs; 59° C.) FreshMaterial Material Heated Sealing Cover Composition (Room Temperature)(24 hrs; 70° C.) Control  0% Loss  0% Loss (No COC, glue, orcrosslinker) COC/Glue/0% crosslinker  0% Loss  0% Loss COC/Glue/0.5%crosslinker 87% Loss 76% Loss COC/Glue/1% crosslinker 86% Loss 12.5%Loss   COC/Glue/3% crosslinker 55% Loss  0% Loss COC/Glue/5% crosslinker97% Loss 95% Loss

In some embodiments, kits are provided, comprising, for example, asealing cover 80 and one or more compatible detection probes that arecompatible (e.g., emission intensity does not degrade when in contact)with sealing cover 80. In some embodiments, a kit can comprise one ormore detection probes that are compatible (e.g., do not degrade overtime when in contact) with adhesive 88 of sealing cover 80. Kits maycomprise a group of detection probes that are compatible with sealingcover 80 comprising adhesive 88 and microplate 20. In some embodiments,the present teachings include methods for matching a group of detectionprobes that are compatible with sealing cover 80 and spotting into atleast some of plurality of wells 26 of microplate 20.

Microplate Sealing Cover Roll

As can be seen in FIGS. 181 and 182, in some of the embodiments, sealingcover 80 can be configured as a roll 512. The use of sealing cover roll512 can provide, in some embodiments, and circumstances, improved easein storage and application of sealing cover 80 on microplate 20 whenused in conjunction with a manual or automated sealing cover applicationdevice, as discussed herein. In some embodiments, sealing cover roll 512can be manufactured using a laminate comprising a protective liner 514,a base stock 516, an adhesive 518, and/or a carrier liner 520. Duringmanufacturing, protective liner 514 can be removed and discarded. Basestock 516 and adhesive 518 can then be kiss-cut, such that base stock516 and adhesive 518 are cut to a desired shape of sealing cover 80, yetcarrier liner 520 is not cut. Excess portions of base stock 516 andadhesive 518 can then be removed and discarded. In some embodiments,base stock 516 can be a scuff resistant and water impermeable layer withlow to no fluorescence.

In some embodiments, carrier liner 520 can then be punched or otherwisecut to a desired shape and finally the combination of carrier liner 520,base stock 516, and adhesive 518 can be rolled about a roll core 522(see FIG. 182). Roll core 522 can be sized so as not to exceed theelastic limitations of base stock 516, adhesive 518, and/or carrierliner 520. In some embodiments, adhesive 518 is sufficient to retainbase stock 516 to carrier liner 520, yet permit base stock 516 andadhesive 518 to be released from carrier liner 520 when desired. In someembodiments, base stock 516, adhesive 518, and carrier liner 520 arerolled upon roll core 522 such that base stock 516 and adhesive 518 facetoward roll core 522 to protect base stock 516 and adhesive 518 fromcontamination and reduce the possibility of premature release.

As can be seen in FIG. 182, in some embodiments, such a desired shape ofcarrier liner 520 can comprise a plurality of drive notches 524 formedalong and slightly inboard of at least one of the elongated edges 526.The plurality of drive notches 524 can be shaped, sized, and spaced topermit cooperative engagement with a drive member to positively drivesealing cover roll 512 and aid in the proper positioning of sealingcover 80 relative to microplate 20. In the some embodiments, the desiredshape of carrier liner 520 can further comprise a plurality of stagingnotches 528 to be used to permit reliable positioning of sealing cover80. In some embodiments, the plurality of staging notches 528 can beformed along at least one elongated edge 526. In some embodiments, theplurality of staging notches 528 can be shaped and sized to permitdetection by a detector, such as an optical detector, mechanicaldetector, or the like. An end/start of roll notch or other feature 530can further be used in some embodiments to provide notification of afirst and/or last sealing cover 80 on sealing cover roll 512. Similar tothe plurality of staging notches 528, end/start of roll notch 530 can beshaped and sized to permit detection by a detector, such as an opticaldetector, mechanical detector, or the like. It should be appreciatedthat the foregoing notches and features can have other shapes than thoseset forth herein or illustrated in the attached figures. It should alsobe appreciated that other features, such as magnetic markers,non-destructive markers (e.g. optical and/or readable markers), or anyother indicia may be used on carrier liner 520. To facilitate suchdetection with an optical detector to avoid physical contact, in someembodiments, carrier liner 520 can be opaque. However, in someembodiments, carrier liner 520 can be generally opaque only nearelongated edges 526 with generally clear center sections 532 to aid inin-process adhesive inspection.

Sealing Cover Applicator

In some embodiments, sealing cover 80 can be laminated onto microplate20 using a hot roller apparatus 540, as illustrated in FIG. 29. In someembodiments, hot roller apparatus 540 comprises a heated top roller 542heated by a heating element 544 and an unheated bottom roller 546. Afirst plate guide 548 can be provided for guiding microplate 20 into hotroller apparatus 540, while similarly a second plate guide 550 can beprovided for guiding microplate 20 out of hot roller apparatus 540.

During sealing, sealing cover 80 can be placed on top of microplate 20and the combination can be fed into hot roller apparatus 540 such thatsealing cover 80 is in contact with first plate guide 548. As sealingcover 80 and microplate 20 pass and engage heated top roller 542, heatcan be applied to sealing cover 80 to laminate sealing cover 80 tomicroplate 20. This laminated combination can then exit hot rollerapparatus 540 as it passes second plate guide 550. In some embodiments,the heat from heated top roller 542 reduces the viscosity of theadhesive of sealing cover 80 to allow the adhesive to better adhere tomicroplate 20.

In some embodiments, hot roller apparatus 540 can variably control theamount of heat applied to sealing cover 80. In this regard, sufficientheat can be supplied to provide adhesive flow or softening of theadhesive of sealing cover 80 without damaging assay 1000. In someembodiments, hot roller apparatus 540 can variably control a drive speedof heated top roller 542 and unheated bottom roller 546. In someembodiments, hot roller apparatus 540 can variably control a clampingforce between heated top roller 542 and unheated bottom roller 546. Byvarying these parameters, optimal sealing of sealing cover 80 tomicroplate 20 can be achieved with minimal negative effects to assay1000.

Manual Sealing Cover Applicator

In some embodiments, sealing cover 80 can be laminated onto microplate20 using a manual sealing cover applicator 552, such as illustrated inFIG. 183. In some embodiments, manual sealing cover applicator 552 canbe used in conjunction with a fixture 554, such as illustrated in FIG.184. In some embodiments, fixture 554 can comprise a generally planarsubstrate 556 comprising a recessed portion 558. Recessed portion 558,in some embodiments, can be longitudinally aligned with generally planarsubstrate 556 and sized to receive microplate 20 therein. In someembodiments, fixture 554 can comprise an alignment feature 560 that canbe complementary to alignment feature 58 on microplate 20. In someembodiments, alignment feature 560 can comprise a corner chamfer, a pin,a slot, a cut corner, an indentation, a graphic, a nub, a protrusion,and/or other unique feature that can be capable of interfacing withalignment feature 58 or other feature of microplate 20. In someembodiments, fixture 554 can comprise one or more recesses 562 formed ingenerally planar substrate 556 to permit, among other things, improvedgrasping of microplate 20 for ease of insertion and withdrawal ofmicroplate 20 from fixture 554. In some embodiments, one or morerecesses 562 can be positioned along opposing ends of microplate 20.

Referring now to FIGS. 183 and 185-187, in some embodiments, manualsealing cover applicator 552 comprises a hinged housing 564 sized toreceive sealing cover roll 512 therein. In some embodiments, hingedhousing 564 comprises a base section 566 and at least one cover section568. In some embodiments, at least one cover section 568 can bepivotally coupled to base section 566 about axis 570. In someembodiments, at least one cover section 568 comprises a pair ofapertures 572 (only one illustrated) formed in sidewalls 574 that caneach be sized to receive a pin 576 extending from an applicator roller578 to permit pivotal movement of at least one cover section 568relative to base section 566. In some embodiments, a latch member 580can be used to releasably couple base section 566 to at least one coversection 568. Latch member 580 can be pivotally coupled to one of basesection 566 and at least one cover section 568 and positionable in alocked position (FIG. 186), coupling base section 566 and at least onecover section 568, and an unlocked position (FIG. 187), permittingrelative pivotal movement of base section 566 and at least one coversection 568.

As illustrated in FIGS. 185-187, in some embodiments, base section 566comprises at least one of applicator roller 578, a support structure582, a roll hub 584, a stretcher 586, a plane assembly 588, anintermediate roller 590, a drive roller assembly 592, a pressure roller594, and a waste gate 596. In some embodiments, applicator roller 578can comprise a generally cylindrical member comprising the pair of pins576 disposed on opposing ends thereof along axis 570. In someembodiments, the pair of pins 576 can engage support structure 582 topermit rotating movement of applicator roller 578 relative thereto. Insome embodiments, applicator roller 578 can be made of, at least inpart, a compliant material to permit applicator roller 578 toaccommodate variations in fixture 554 and/or microplate 20.

In some embodiments, roll hub 584 can be fixedly coupled to supportstructure 582 to support sealing cover roll 512 thereon and permitrelative rotation therebetween. In some embodiments, roll hub 584comprises a pair of friction legs 598 extending outwardly fromtangential sections 600 of a central portion 602. In some embodiments,the pair of friction legs 598 can each extend along only a portion ofroll hub 584. The pair of friction legs 598 can be sized to frictionallyengage an inner surface of roll core 522 of sealing cover roll 512 toprovide drag and/or positively retain sealing cover roll 512 on roll hub584.

In some embodiments, stretcher 586 comprises a bracket portion 604 andan engaging portion 606. In some embodiments, bracket portion 604 can befixedly coupled to support structure 582 to provide a generally rigidsupport. In some embodiments, engaging portion 606 comprises a mountingsection 608 and one or more finger members 610 extending from mountingsection 608. The one or more finger members 610 can comprise an upturnedend 612 to form an engaging corner 614 to contact sealing cover roll 512as it passes thereby. In some embodiments, mounting section 608 can befixedly coupled to bracket portion 604 via conventional fasteners and/ora tab member interface 616 (FIG. 185).

Still referring to FIGS. 185-187, in some embodiments, plane assembly588 comprises a plate member 618 and a plane roller 620 rotatablycoupled to plate member 618 along axis 622. In some embodiments, planeroller 620 can be a generally cylindrical member comprising a pair ofpins 624 disposed on opposing ends thereof along axis 622. In someembodiments, the pair of pins 624 can engage apertures formed in platemember 618 to permit rotating movement of plane roller 620 relativethereto. In some embodiments, plane roller 620 can be made of, at leastin part, a compliant material to permit plane roller 620 accommodatevariations in fixture 554 and/or microplate 20. In some embodiments,plane roller 620 can carry carrier liner 520 of sealing cover roll 512.In some embodiments, plane roller 620 can be sized to apply a force on abackside of carrier liner 520 and, consequently, on sealing cover 80 toadhere sealing cover 80 to microplate 20 during application. In someembodiments, carrier liner 520 can then travel along plate member 618 tointermediate roller 590. It should be appreciated that plane roller 620can comprise posts (not illustrated) formed thereon to engage theplurality of drive notches 524 formed on some embodiments of carrierliner 520 to aid in alignment.

In some embodiments, intermediate roller 590 can comprise a generallycylindrical member comprising a pair of pins 626 disposed on opposingends thereof along axis 628. In some embodiments, the pair of pins 626can engage apertures formed in support structure 582 to permit rotatingmovement of intermediate roller 590 relative thereto. In someembodiments, intermediate roller 590 can be comprises of, at least inpart, a compliant material to permit intermediate roller 590 toaccommodate variations in fixture 554 and/or microplate 20. In someembodiments, intermediate roller 590 can carry carrier liner 520 ofsealing cover roll 512. In some embodiments, intermediate roller 590 canbe tapered along its longitudinal length to a reduced cross-section areaat about a longitudinal midpoint of intermediate roller 590. Thistapered configuration can aid in maintaining carrier liner 520 generallycentered on intermediate roller 590. In some embodiments, intermediateroller 590 can be sized to apply a force on a backside of carrier liner520 and, consequently, on sealing cover 80 to adhere sealing cover 80 tomicroplate 20 during application.

As best seen in FIG. 185, in some embodiments, drive roller assembly 592comprises at least one knob portion 630 disposed on at least one end ofa drive roller 632. In some embodiments, drive roller 632 can comprise agenerally cylindrical member comprising a pair of pins 634 (illustratedhidden in FIG. 185) disposed on opposing ends thereof along axis 636. Insome embodiments, the pair of pins 634 can engage apertures formed insupport structure 582 to permit rotating movement of drive roller 632relative thereto. In some embodiments, the pair of pins 634 can furtherengage the at least one knob portion 630. In some embodiments, a pair ofknob portions 630 can be used and disposed on opposing ends of driveroller 632 to permit both left-handed and right-handed operation. Knobportion 630 can be manually manipulated by a user to manually advancecarrier liner 520 of sealing cover roll 512. In some embodiments, driveroller 632 can be comprised of, at least in part, a compliant materialto permit drive roller 632 to accommodate variations in fixture 554and/or microplate 20. In some embodiments, drive roller 632 can be sizedto apply a force on a backside of carrier liner 520 and, consequently,on sealing cover 80 to adhere sealing cover 80 to microplate 20 duringapplication.

In some embodiments, drive roller 632 can be sized to operably engagepressure roller 594 to receive carrier liner 520 of sealing cover roll512 therebetween (see FIG. 185). In some embodiments, pressure roller594 can be a generally cylindrical member comprising a pair of pins 638disposed on opposing ends thereof along axis 640. In some embodiments,the pair of pins 638 can engage apertures formed in a support bracket642 to permit rotating movement of pressure roller 594 relative thereto.In some embodiments, support bracket 642 can be fixedly mounted to orintegrally formed with at least one cover section 568. In someembodiments, pressure roller 594 can be biased to apply a force againstdrive roller 632 to, at least in part, positively grab, and/or advancecarrier liner 520.

Finally, in some embodiments, carrier liner 520 of sealing cover roll512 can be fed from a lower portion of sealing cover roll 512 forwardalong a top side of plate member 618. Carrier liner 520 can then be fedaround plane roller 620, along an bottom side of plate member 618,around intermediate roller 590, between pressure roller 594 and driveroller 632, and finally out of waste gate 596.

In some embodiments, during operation, a user can manually manipulate atleast one knob portion 630 until an edge of sealing cover 80 can beadvanced to a predetermined seal position. In some embodiments, manualsealing cover applicator 552 can then be placed on top of fixture 554having microplate 20 mounted thereon. In some embodiments, the user canthen apply a downward force on, at least in part, a handle member 640and push/pull manual sealing cover applicator 552 from one end ofmicroplate 20 to an opposing end of microplate 20. This motion and theconstruction of manual sealing cover applicator 552 causes sealing cover80 to engage and be mounted to microplate 20. In some embodiments, thedownward force applied to manual sealing cover applicator 552 activatesadhesive 518. This motion, in some embodiments, serves to expel thewaste (i.e. carrier liner 520 having no sealing cover 80) out of wastegate 596.

In some embodiments, sealing cover roll 512 can be loaded in manualsealing cover applicator 552 by positioning latch member 580 in theunlocked position (FIG. 187) and pivoting at least one cover section 568upward. Sealing cover roll 512 can then be place on roll hub 584.Carrier liner 520 can then be routed through manual sealing coverapplicator 552 as described above.). In some embodiments, closing of theat least one cover section 568 causes pressure roller 594 to apply aforce on carrier liner 520. In some embodiments, drive roller 632 and/orknob section 630 can be ratcheted to maintain carrier liner 520 undertension.

It should be appreciated that this arrangement can provide reducedpossibility of sealing cover application defects, improved sealing coverplacement accuracy, reduced operator skill, and faster sealing coverapplication.

Automated Sealing Cover Applicator—Roll

In some embodiments, as illustrated in FIGS. 188-192, sealing cover 80can be laminated onto microplate 20 using an automated sealing coverapplicator 1100. In some embodiments, automated sealing cover applicator1100 comprises a housing 1102 sized to receive sealing cover roll 512therein. In some embodiments, housing 1102 can comprise a base section1104 and cover section 1106 connectable therewith. In some embodiments,cover section 1106 can comprise an opening 1108 for receiving a sealingcover cassette 1110 therein.

Referring now to FIGS. 189 and 190, in some embodiments, base section1104 comprises at least one of a microplate tray assembly 1112, a traydrive system 1114, a sealing cover drive system 1116 for at least inpart alignment control of sealing cover roll 512, a heated rollerassembly 1118, and an applicator control system 1120.

In some embodiments, microplate tray assembly 1112 comprises a generallyplanar tray member 1122 that can be movable between an extended position(FIGS. 188-190) and a retracted position. In some embodiments, generallyplanar tray member 1122 comprises a recessed portion 1124. Recessedportion 1124, in some embodiments, can be sized to receive microplate 20therein. In some embodiments, microplate tray assembly 1112 comprises analignment feature 1126 that can be complementary to alignment feature 58on microplate 20. In some embodiments, alignment feature 1126 can acorner chamfer, a pin, a slot, a cut corner, an indentation, a graphic,a nub, a protrusion, or other unique feature that can be capable ofinterfacing with alignment feature 58 or other feature of microplate 20.In some embodiments, microplate tray assembly 1112 comprises one or morerecesses 1128 formed in generally planar tray member 1122 to permit,among other things, improved grasping of microplate 20 for ease ofinsertion and withdrawal of microplate 20 from microplate tray assembly1112. In some embodiments, one or more recesses 1128 can be positionedalong opposing ends of microplate 20. In some embodiments, generallyplanar tray member 1122 comprises a uniquely sized and/or shaped insert1130 that can be fastened within recessed portion 1124 to accommodatevarying sizes of microplates or other devices.

As can be seen in FIG. 190, in some embodiments, microplate trayassembly 1112 can be moved between the extended position and theretracted position via tray drive system 1114. In some embodiments, traydrive system 1114 comprises at least one of a drive motor 1132 and adrive track member 1134. In some embodiments, drive track member 1134can be a threaded member, such as but not limited to a worm gear,threadedly engaging a receiver 1136 fixedly coupled to microplate trayassembly 1112. Drive motor 1132 can be actuated by a control switchand/or applicator control system 1120 to rotatably turn drive trackmember 1134. In turn, microplate tray assembly 1112 can travel relativeto drive track member 1134 between the extended and retracted positions.During such travel, microplate tray assembly 1112 can be guided via atleast one guide member 1137 mounted within base section 1104. It shouldbe appreciated that tray drive system 1114 comprises a cable drivesystem, a track drive system, a rack and pinion system, a hydraulicsystem, a pneumatic system, a solenoid system, or the like.

In some embodiments, as illustrated in FIGS. 189-192, sealing covercassette 1110 comprises at least one of a support structure 1138, acover member 1140, a roll hub 1142, a plane roller 1144, at least onefeed roller 1146, a sprocket drive member 1148, and a waste gate 1150.

In some embodiments, roll hub 1142 can be fixedly coupled to supportstructure 1138 to support sealing cover roll 512 thereon and permitrelative rotation therebetween. In some embodiments, roll hub 1142comprises pair of friction legs 598 extending outwardly from tangentialsections 600 of central portion 602 as discussed herein. In someembodiments, roll hub 1142 can comprise a cylindrical support member1152.

In some embodiments, plane roller 1144 can be a generally cylindricalmember rotatably supported by support structure 1138 to permit rotatingmovement of plane roller 1144 relative thereto. In some embodiments,plane roller 1144 can be made of, at least in part, a compliant materialto permit plane roller 1144 to accommodate variations in microplate trayassembly 1112 and/or microplate 20. In some embodiments, plane roller1144 can be sized and/or positioned to engage microplate tray assembly1112 and/or microplate 20 to apply a compressing force upon sealingcover 80 and microplate 20 to impart at least an initial sealingengagement.

In some embodiments, the at least one feed roller 1146 can comprise apair of cylindrical members rotatably supported by support structure1138 to permit rotating movement of feed roller 1146 relative thereto.In some embodiments, feed rollers 1146 can be made of a material to, atleast in part, positively grab and/or advance carrier liner 520. Feedroller 1146 can also be configured to impart a drag force on carrierliner 520 opposing a driving force by sprocket drive member 1148 toensure carrier liner 520 and sealing cover 80 disposed thereon aregenerally flat between feed roller 1146 and sprocket drive member 1148.

As best seen in FIG. 185, in some embodiments, sprocket drive member1148 can be a generally cylindrical member comprising at least onesprocket portion 1154 disposed on at least one end of a support rod 1156(FIG. 189) rotatable about an axis 1157. In some embodiments, a pair ofsprocket portions 1154 can be provided such that each of the pair ofsprocket portions 1154 can be disposed on opposing ends of support rod1156. In some embodiments, support rod 1156 can be rotatably coupled tosupport structure 1138. The pair of sprocket portions 1154 can eachcomprise a plurality of engaging portions 1158 that are each sized andspaced to enmesh with each of the plurality of drive notched 524 formedon carrier liner 520 of sealing cover roll 512.

In some embodiments, sprocket drive member 1148 can be driven by sealingcover drive system 1116. In some embodiments, sealing cover drive system1116 can comprise a drive motor 1160 (FIG. 189) enmeshingly engaging adrive gear 1162 (FIG. 191) fixed coupled at an end of support rod 1156of sprocket drive member 1148 (FIG. 191). In some embodiments, drivemotor 1160 can be actuated by a control switch and/or applicator controlsystem 1120 to rotatably turn sprocket drive member 1148 and drivecarrier liner 520 of sealing cover roll 512. In some embodiments, drivemotor 1160 can be fixedly mounted within base section 1104. In someembodiments, a vibration isolation member 1164 can be disposed betweendrive motor 1160 and a support structure 1166 within base section 1104.

As best seen in FIG. 192, in some embodiments, carrier liner 520 ofsealing cover roll 512 can be fed from sealing cover roll 512 downwardbetween feed roller 1146 and around sprocket drive members 1148 and outwaste gate 1150. To aid in initial feeding of carrier liner 520 aroundsprocket drive members 1148, a guide wall 1168 can be provided to directan end of carrier liner 520 toward waste gate 1150.

In some embodiments, as illustrated in FIGS. 190 and 192, sealing covercassette 1110 can further comprise a latch system 1170 for operablycoupling sealing cover cassette 1110 to cover section 1106. In someembodiments, latch system 1170 comprises a lip member 1172 disposed onone end of cover member 1140 and at least one biasing members 1174. Asbest seen in FIG. 192, lip member 1172 can engage an underside of coversection 1106. Similarly, at least one biasing member 1174 can begenerally U-shaped and have a retaining feature 1177 that can be sizedto engage an underside of cover section 1106. In this regarding, atleast one biasing member 1174 can impart a locking force such thatretaining feature 1177 remains engaged with the underside of coversection 1106 until a user overcomes the biasing force to disengageretaining feature 1177 from cover section 1106. To install sealing covercassette 1110 into cover section 1106, one can simply insert lip member1172 under cover section 1106 and pivot a front end of sealing covercassette 1110 downward until the at least one biasing member 1174engages cover section 1106. This motion can further engage drive gear1162 with drive motor 1160.

As illustrated in FIG. 190, in some embodiments, heated roller assembly1118 can be used to apply at least one of heat and pressure to sealingcover 80 and/or microplate 20 as tray generally planar tray member 1122passed therebelow. In some embodiments, heat and/or pressure can be usedto activate adhesive 518 on sealing cover 80 to effect sealing interface112. In some embodiments, heated roller assembly 1118 comprises a heatedroller 1178 rotatably supported within a removable housing 1180. In someembodiments, heated roller 1178 can be heated internally via a heatingmember 1182 and/or heated externally via a heating device 1184. In someembodiments, heating member 1182 and/or heating device 1184 can becontrolled by applicator control system 1120. It should be appreciatedthat heated roller assembly 1118 can be manufactured as a sub-assemblyto permit easy retrofitting of existing automated sealing coverapplicators 1100 for use with heat sensitive adhesives. It should alsobe appreciated that in some embodiments, heating device 1184 can serveas a convective and/or indirect heater of sealing cover 80 as microplate20 passes therebelow. In such embodiments, heated roller 1178 can beeliminated.

In some embodiments, applicator control system 1120 can be operable tocontrol tray drive system 1114 and/or sealing cover drive system 1116 toapply sealing cover 80 to microplate 20. Applicator control system 1120comprises an electrical circuit operable to output various controlsignals to drive motor 1132 and/or drive motor 1160 in response to aprogram mode of operation and/or data input. In some embodiments,applicator control system 1120 can receive data input from at least onesensor disposed in automated sealing cover applicator 1100, such as, butnot limited to, a tray drive sensor for detecting encumbered operationof microplate tray assembly 1112, a sealing cover drive sensor fordetecting encumbered operation of sealing cover cassette 1110, a sealingcover position sensor for detecting one of the plurality of stagingnotches 528 formed in carrier liner 520, an end/start of roll sensor fordetecting end/start of roll notch 530, a temperature sensor fordetecting a temperature of heated roller 1178, or any other sensor fordetecting a desired operating parameter of automated sealing coverapplicator 1100. In some embodiments, applicator control system 1120 canbe response to at least one of a power switch 1186, a tray activationbutton 1188, and/or a seal application button 1190 (FIG. 188). Stillfurther, in some embodiments, applicator control system 1120 can outputa control status indicia 1192 that can include, but is not limited to, aTEMP alert indicia, a SEAL EMPTY alert indicia, a TRAY JAM alertindicia, a SEAL JAM alert indicia, a POWER alert indicia, a READY alertindicia, or the like. In some embodiments, the TEMP alert indicia can beused to indicate when a desired temperature has been reached. In someembodiments, the SEAL EMPTY alert indicia can be used to indicate whensealing cover roll 512 is at or near empty of sealing covers 80. In someembodiments, the TRAY JAM alert indicia can be used to indicate whenmicroplate tray assembly 1112 is encumbered. In some embodiments, theSEAL JAM alert indicia can be used to indicate when at least one sealingcover 80 is encumbered.

It should be appreciated that this arrangement can provide reducedpossibility of sealing cover application defects, improved sealing coverplacement accuracy, reduced operator skill, and faster sealing coverapplication.

Automated Sealing Cover Applicator—Single Sheet

Turning now to FIGS. 193-201, in some embodiments, automated sealingcover applicator 1100 comprises a single sheet applicator assembly 1194.In some embodiments, single sheet applicator assembly 1194 comprises atleast one of a plate member 1196, a cartridge receiving assembly 1198, asealing cover cartridge 1200, and a planer drive system 1202.

As can be seen in FIGS. 195 and 197, in some embodiments, sealing covercartridge 1200 comprises at least one of a top cover 1204, a bottomcover 1206, a separator 1208, at least one wheel member 1210, and asealing cover carrier assembly 1212. In some embodiments, sealing covercarrier assembly 1212 comprises a carrier liner 1214 and a sealing cover80 disposed on carrier liner 1214. In some embodiments, carrier liner1214 can be sized larger than sealing cover 80 to define a flap 1216along a leading edge of carrier liner 1214. In some embodiments, carrierliner 1214 can be similar in material to carrier liner 520.

In some embodiments, top cover 1204 can be generally planar inconstruction and comprises a pair of feed slots 1218 formed along aleading edge 1220 thereof. The pair of feed slots 1218 can be sized toreveal a portion of sealing cover carrier assembly 1212, specificallyflap 1216, for later use in dispensing sealing cover 80.

In some embodiments, bottom cover 1206 can be generally planar inconstruction and can comprise a pair of feed slots 1222 formed along aleading edge 1224 thereof. The pair of feed slots 1222 can be sized togenerally align with the pair of feed slots 1218 of top cover 1204 toreveal a portion of sealing cover carrier assembly 1212, specificallyflap 1216, for later use in dispensing sealing cover 80.

In some embodiments, separator 1208 can be generally planar inconstruction and can be sized to be generally received within top cover1204 and bottom cover 1206. In some embodiments, separator 1208 cancomprise at least one rib 1226 extending about a periphery of separator1208 and/or traversing thereabout to support sealing cover carrierassembly 1212 thereon. Separator 1208 can further comprise at least onecoupling member 1228 for retaining at least one wheel member 1210. Insome embodiments, the at least one coupling member 1228 can be aC-shaped members sized to engage and retain a reduced cross-sectionportion 1230 of at least one wheel member 1210. In some embodiments, theouter diameter of the at least one coupling member 1228 can be less thanthe outer diameter the at least one wheel member 1210 to reduceinterference between the at least one coupling member 1228 and sealingcover carrier assembly 1212.

In some embodiments, top cover 1204, separator 1208, and bottom cover1206 can be coupled together to encapsulate sealing cover carrierassembly 1212 and sealing cover 80 therein, as illustrated in FIG. 196.Bottom cover 1206 can comprise at least one mounting stud 1232 formed onan interior side thereof. Top cover 1204 and separator 1208 can compriseat least one aperture 1234 generally aligned with the at least onemounting stud 1232 to receive a threaded fastener therethrough. However,it should be appreciate that other coupling systems, such as a snap-lockinterface, can be used. As illustrated in FIG. 196, in some embodiments,a slot 1236 can be formed between top cover 1204 and bottom cover 1206.Slot 1236 can be generally aligned with a tangent of sealing covercarrier assembly 1212 such that as carrier liner 1214 can be drivenabout the at least one wheel member 1210, sealing cover 80 can beencouraged to delaminate from carrier liner 1214 and be urged fromsealing cover cartridge 1200 for application upon microplate 20.

As best seen in FIGS. 193, 194, and 198-201, in some embodiments,sealing cover 80 can be urged from sealing cover cartridge 1200 forapplication upon microplate 20 by first inserting sealing covercartridge 1200, having sealing cover 80 disposed therein, into cartridgereceiving assembly 1198. In some embodiments, cartridge receivingassembly 1198 comprises a removable cartridge support 1238. Removablecartridge support 1238 can be sized to receive sealing cover cartridge1200 therein for insertion into automated sealing cover applicator 1100.Automated sealing cover applicator 1100 comprises an opening 1240 formedin a cover section 1242. In some embodiments, cover section 1242 canhave an inwardly-extending angled lip portion 1244. Angled lip portion1244 can support and retain an adjustable handle member 1246 via afastener 1247. In some embodiments, adjustable handle member 1246comprises a grasping portion 1248 and an urging member 1250 disposed onan opposing end of adjustable handle member 1246 relative to graspingportion 1248. In some embodiments, urging member 1250 can be operable toengage a backside of removable cartridge support 1238 and urge sealingcover cartridge 1200 toward planer drive system 1202.

In some embodiments, planer drive system 1202 comprises a generallytriangular mounting block 1252 and at least one drive roller 1254mounted thereto that can be sized and generally aligned with at leastone feed slot 1218, 1222 to operably engage flap 1216 of carrier liner1214 to drive sealing cover carrier assembly 1212 and urge sealing cover80 out of slot 1236. In some embodiments, at least one drive roller 1254can be operably driven via a drive motor, such as drive motor 1160,through a gear assembly 1256 (FIG. 194).

With particular reference to FIGS. 198-201, planer drive system 1202 canfurther comprise a plane roller 1258. In some embodiments, plane roller1258 can be a generally cylindrical member rotatably supported bysupport structure 1166 to permit rotating movement of plane roller 1258relative thereto. In some embodiments, plane roller 1258 can be made of,at least in part, a compliant material to permit plane roller 1258 toaccommodate variations in microplate tray assembly 1112 and/ormicroplate 20. In some embodiments, plane roller 1258 can be sizedand/or positioned to engage microplate tray assembly 1112 and/ormicroplate 20 to apply a compressing force upon sealing cover 80 andmicroplate 20 to impart at least an initial sealing engagement. In someembodiments, plane roller 1258 can be heated.

During operation, in some embodiments, sealing cover carrier assembly1212, carrying a single sealing cover 80, can be preloaded or loaded bya user into sealing cover cartridge 1200 such that flap 1216 of carrierliner 1214 can be exposed through at least one feed slot 1218, 1222.This arrangement can provide reduced contamination of sealing cover 80and microplate 20. As illustrated in FIG. 198, sealing cover cartridge1200 can then be loaded into removable cartridge support 1238 andinserted into opening 1240 of cover section 1242 until urging member1250 engages removable cartridge support 1238 such that flap 1216 can beurged against at least one drive roller 1254 of planer drive system1202. Microplate 20 can be loaded into microplate tray assembly 1112. Asillustrated in FIG. 199, microplate tray assembly 1112 can then beeither manually or automatically driven into automated sealing coverapplicator 1100. At least one drive roller 1254 can then be actuated ata predetermined time to drive flap 1216 of carrier liner 1214 about atleast one wheel member 1210. However, because of, at least in part, theradius of the at least one wheel member 1210, sealing cover 80 can bedelaminated from carrier liner 1214 and urged out of slot 1236, asillustrated in FIG. 200. Finally, sealing cover 80 can generally engagemicroplate 20 and plane roller 1258 applies a compressing force uponsealing cover 80 and microplate 20 to impart at least an initial sealingengagement between sealing cover 80 and microplate 20. This arrangementcan provide reduced possibility of sealing cover application defects,improved sealing cover placement accuracy, reduced operator skill, andfaster sealing cover application.

Thermocycler System

With reference to FIGS. 30-44, 47, and 48, in some embodiments,thermocycler system 100 comprises at least one thermocycler block 102.Thermocycler system 100 provides heat transfer between thermocyclerblock 102 and microplate 20 during analysis to vary the temperature of asample to be processed. It should be appreciated that in someembodiments thermocycler block 102 can also provide thermal uniformityacross microplate 20 to facilitate accurate and precise quantificationof an amplification reaction. In some embodiments, a control system 1010(FIGS. 30, 41, and 42) can be operably coupled to thermocycler block 102to output a control signal to regulate a desired thermal output ofthermocycler block 102. In some embodiments, the control signal ofcontrol system 1010 can be varied in response to an input from atemperature sensor (not illustrated).

In some embodiments, thermocycler block 102 comprises a plurality of finmembers 104 (FIGS. 42 and 44) disposed along a side thereof to dissipateheat. In some embodiments, thermocycler block 102 comprises at least oneof a forced convection temperature system that blows hot and cool aironto microplate 20; a system for circulating heated and/or cooled gas orfluid through channels in microplate 20; a Peltier thermoelectricdevice; a refrigerator; a microwave heating device; an infrared heater;or any combination thereof. In some embodiments, thermocycler system 100comprises a heating or cooling source in thermal connection with a heatsink. In some embodiments, the heat sink can be configured to be inthermal communication with microplate 20. In some embodiments,thermocycler block 102 continuously cycles the temperature of microplate20. In some embodiments, thermocycler block 102 cycles and then holdsthe temperature for a predetermined amount of time. In some embodiments,thermocycler block 102 maintains a generally constant temperature forperforming isothermal reactions upon or within microplate 20.

Multiple Thermocyclers

In some embodiments, a plurality of thermocycler blocks 102 can beemployed to thermally cycle a plurality of microplates 20 to permithigher throughput of microplates 20 through high-density sequencedetection system 10. In some embodiments, each of the plurality ofthermocycler blocks 102 can thermally cycle a separate microplate 20 toincrease the overall duty cycle of detection system 300 and, in turn,high-density sequence detection system 10. In other words, during atypical PCR analysis, temperature cycles are used, at least in part, todenature (at a high temperature, e.g, about 95° C.) and then extend (ata low temperature, e.g., about 60° C.) a DNA target. Conventionaldetection systems can then measure a resultant emission while at the lowtemperature. However, as can be appreciated, during these temperaturecycles, conventional detection systems are idle until the next lowtemperature portion of the cycle. For instance, in cases where about 40temperature cycles are completed over a 2-hour period, the conventionaldetection system is active to measure the resultant emission about 40times. The remaining time the conventional detection system is idle.Therefore, it should be appreciated that conventional thermocyclersystems limit the duty cycle of conventional excitation systems and/orconventional detection systems.

In some embodiments, for example, the plurality of thermocycler blocks102 can be synchronized to provide offset temperature cycles. In someembodiments, the plurality of thermocycler blocks 102 can besynchronized to maximize or provide at or near 100% usage of detectionsystem 300. The exact number of thermocycler blocks 102 to be used is,at least in part, dependent on the time required to measure all thesamples on a single thermocycler and the degree of time offset betweenthe cycling profiles of each thermocycler system.

In some embodiments, detection system 300 can comprise a driving deviceto position detection system 300 and, in some embodiments, excitationsystem 200 above one of the plurality of thermocycler blocks 102 tomeasure a resultant emission from the corresponding microplate 20. Insome embodiments, detection system 300 can comprise a movable mirror topermit measurement of the resultant emission of multiple microplates 20from a fixed position. In some embodiments, each of the plurality ofthermocycler blocks 102 can be positioned on a carousel or track systemfor movement relative to detection system 300. It should be appreciatedthat any system, in addition to those described herein, can be used topermit detection of resultant emission from one or more microplates 20positioned on the plurality of thermocycler blocks 102 by a singledetection system 300 to increase the duty cycle thereof.

Thermal Compliant Pad

With reference to FIG. 33, thermal compliant pad 140 can be disposedbetween thermocycler block 102 and any adjacent component, such asmicroplate 20 or a sealing cover 80. It should be understood thatthermal compliant pad 140 is optional. Thermal compliant pad 140 canbetter distribute heating or cooling through a contact interface betweenthermocycler block 102 and the adjacent component. This arrangement canreduce localized hot spots and compensate for surface variations inthermocycler block 102, thereby providing improved thermal distributionacross microplate 20.

Pressure Clamp System

As will be further described herein, according to some embodiments,pressure clamp system 110 can apply a clamping force upon sealing cover80, microplate 20, and thermocycler block 102 to, at least in part,operably seal assay 1000 within the plurality of wells 26 duringthermocycling and further improve thermal communication betweenmicroplate 20 and thermocycler block 102. Pressure clamp system 110 canbe configured in any one of a number of orientations, such as describedherein. Additionally, pressure clamp system 110 can comprise any one ofa number of components depending upon the specific orientation used.Therefore, it should be understood that variations exist that are stillregarded as being within the scope of the present teachings.

Transparent Bag

As illustrated in FIGS. 30-33, in some embodiments, pressure clampsystem 110 can comprise an inflatable transparent bag 116 positionedbetween and in engaging contact with a transparent window 112 andsealing cover 80. In the embodiment illustrated in FIG. 30, transparentwindow 112 and thermocycler block 102 are fixed in position againstrelative movement. Inflatable transparent bag 116 comprises aninflation/deflation port 118 that can be fluidly coupled to a pressuresource 122, such as an air cylinder, which can be controllable inresponse to a control input from a user or control system 1010. Itshould be understood that in some embodiments inflatable transparent bag116 can comprise a plurality of inflation/deflation ports to facilitateinflation/deflation thereof.

Upon actuation of pressure source 122, pressurized fluid, such as air,can be introduced into inflatable transparent bag 116, thereby inflatingtransparent bag 116 in order to exert a generally uniform force upontransparent window 112 and upon sealing cover 80 and microplate 20. Insome embodiments, such generally uniform force can serve to provide areliable and consistent sealing engagement between sealing cover 80 andmicroplate 20. This sealing engagement can substantially prevent waterevaporation or contamination of assay 1000 during thermocycling. In someembodiments, inflatable transparent bag 116 can be part of thetransparent window 112, thereby forming a bladder.

Still referring to FIG. 30, it should be appreciated that in someembodiments transparent window 112, inflatable transparent bag 116, andsealing cover 80 permit free transmission therethrough of an excitationlight 202 generated by an excitation system 200 and the resultantfluorescence emission. Transparent window 112, inflatable transparentbag 116, and sealing cover 80 can be made of a material that isnon-fluorescent or of low fluorescence. In some embodiments, transparentwindow 112 can be comprised of Vycor®, fused silica, quartz, high purityglass, or combination thereof. By way of non-limiting example, window112 can be comprised of Schott Q2 quartz glass. In some embodiments,window 112 can be from about ¼ to about ½ inch thick; e.g., in someembodiments, about ⅜ inch thick. In some embodiments, a broadbandanti-reflective coating can be applied to one or both sides of window112 to reduce glare and reflections. In some embodiments, thetransparent window 112 can comprise optical elements such as a lens,lenslets, and/or a holographic feature.

In some embodiments, as illustrated in FIG. 31, transparent window 112can be movable to exert a generally uniform force upon transparent bag116 and, additionally, upon sealing cover 80 and microplate 20. In thisembodiment as in others, transparent bag 116 can comprise a fixedinternal amount of fluid, such as air. Transparent window 112 can bemovable using any moving mechanism (not illustrated), such as anelectric drive, mechanical drive, hydraulic drive, or the like.

Pressure Chamber

In some embodiments, as illustrated in FIGS. 34-40, pressure clampsystem 110 can further employ a pressure chamber 150 in place oftransparent bag 116.

Pressure chamber 150 can be a pressurizable volume generally defined bytransparent window 112, a frame 152 that can be coupled to transparentwindow 112, and a circumferential chamber seal 154 disposed along anedge of frame 152. Circumferential chamber seal 154 can be adapted toengage a surface to define the pressurizable, airtight, or at least lowleakage, pressure chamber 150. Transparent window 112, frame 152,circumferential chamber seal 154, and the engaged surface bound theactual volume of pressure chamber 150. Circumferential chamber seal 154can engage one of a number of surfaces that will be further discussedherein. A port 120, in fluid communication with pressure chamber 150 andpressure source 122, can provide fluid to pressure chamber 150.

In the interest of brevity, it should be appreciated that the particularconfiguration and arrangement of sealing cover 80 and microplate 20illustrated in FIGS. 34-40 can be similar to that illustrated in FIGS.30-33.

In some embodiments, as illustrated in FIGS. 34 and 36, circumferentialchamber seal 154 can be positioned such that it engages a portion ofsealing cover 80. A downward force from transparent window 112 can beexerted upon microplate 20 to maintain a proper thermal engagementbetween microplate 20 and thermocycler block 102. Additionally, suchdownward force can further facilitate sealing engagement of sealingcover 80 and microplate 20. Still further, pressure chamber 150 can thenbe pressurized to exert a generally uniform force upon sealing cover 80and sealing interface 92. Such generally uniform force can provide areliable and consistent sealing engagement between sealing cover 80 andmicroplate 20. This sealing engagement can reduce water evaporation orcontamination of assay 1000 during thermocycling.

With particular reference to FIG. 37, it should be appreciated that insome embodiments circumferential chamber seal 154 of pressure chamber150 can be positioned to engage thermocycler block 102, rather thanmicroplate 20. Microplate 20 can be positioned within pressure chamber150. As pressure chamber 150 is pressurized, force is exerted uponsealing cover 80, thereby providing a sealing engagement between sealingcover 80 and microplate 20.

In some embodiments, as illustrated in FIG. 39, to improve thermalcontact between microplate 20 and thermocycler block 102, optional posts156 can be employed. Optional posts 156 can be adapted to be coupledwith transparent window 112 and downwardly extend therefrom. Optionalposts 156 can then engage at least one of microplate 20 or sealing cover80 to ensure proper contact between microplate 20 and thermocycler block102 during thermocycling.

Inverted Orientation

In some embodiments, as illustrated in FIGS. 27, 32, 35, 41, 44, 47, and48, microplate 20 can be inverted such that each of the plurality ofwells 26 is generally inverted, such that the opening of each of theplurality of wells 26 is directed downwardly. Among other things, thisarrangement can provide improved fluorescence detection. As illustratedin FIG. 27, this inverted arrangement causes assay 1000 to collectadjacent sealing cover 80 and, thus, addresses the occurrence ofcondensation effecting fluorescence detection and improves opticalefficiency, because assay 1000 is now disposed adjacent to the openingof each of the plurality of wells 26.

In some embodiments, as illustrated in FIG. 32, thermocycler block 102remains stationary and is positioned above microplate 20 and transparentwindow 112 is positioned below microplate 20. Inflatable transparent bag116 can then be positioned in engaging contact between transparentwindow 112 and sealing cover 80. It should be appreciated thattransparent window 112, inflatable transparent bag 116, and sealingcover 80 can permit free transmission therethrough of excitation light202 generated by excitation system 200 positioned below transparentwindow 112 and the resultant fluorescence therefrom. In someembodiments, detection system 300 can be positioned below microplate 20to detect such fluorescence generated in response to excitation light202 of excitation system 200.

In some embodiments, as illustrated in FIG. 35, microplate 20 can bepositioned in an inverted orientation, similar to that described inconnection with FIG. 32, and further employ pressure chamber 150.Circumferential chamber seal 154 can then be positioned such that itengages a portion of sealing cover 80. A force from transparent window112 can be exerted upon microplate 20 to maintain a proper thermalengagement between microplate 20 and thermocycler block 102 and sealingengagement between sealing cover 80 and microplate 20. Pressure chamber150 can then be pressurized to exert a generally uniform force acrosssealing cover 80.

Vacuum Channels

As illustrated in FIG. 38, some embodiments can comprise a vacuum assistsystem 170. In this regard, in some embodiments, port 120 can beeliminated. Vacuum assist system 170 can comprise a pressure/vacuumsource 172 fluidly coupled to at least one vacuum channel 174, whichextends throughout thermocycler block 102. Vacuum channel 174 cancomprise grooves or, alternatively or in addition, can comprise a porousor permeable section of thermocycler block 102. Vacuum channel 174 canbe evacuated so as to form a vacuum within a volume 176 defined bytransparent window 112, an 0-ring 178, and thermocycler block 102. Uponactuation of pressure source 172, a vacuum can be formed in vacuumchannel 174. This vacuum can vacate volume 176 causing outside airpressure to exert a clamping force on transparent window 112, therebyclamping sealing cover 80 against microplate 20 to ensure a proper sealand further clamping microplate 20 to thermocycler block 102 to ensure aproper thermal contact. It should be understood that in some embodimentsvacuum assist system 170 can be formed in transparent window 112.

Relief Port

Turning now to FIG. 40, in some embodiments a relief port 158 can be influid communication with pressure chamber 150. Relief port 158 can beoperable to slowly bleed gas in pressure chamber 150 and/orsimultaneously remove water vapor from pressure chamber 150 to reducecondensation. Removal of water vapor can, in some circumstances, improvefluorescence detection. Relief port 158 can be used in connection withany of the embodiments described herein.

Window Heating Device

In some embodiments, as illustrated in FIG. 41, transparent window 112can comprise a heating device 160. Heating device 160 can be operable toheat transparent window 112, which in turn heats each of the pluralityof wells 26 to reduce the formation of condensation within each of theplurality of wells 26. In some cases, condensation can reduce opticalperformance and, thus, reduce the efficiency and/or stability offluorescence detection.

In some embodiments, heating device 160 can comprise a layer member 162that can be laminated to transparent window 112. In some embodiments,layer member 162 can comprise a plurality of heating wires (notillustrated) distributed uniformly throughout layer member 162, whichcan each be operable to heat an adjacent area. In some embodiments,layer member 162 can be an indium tin oxide coating that is applieduniformly across transparent window 112. A pair of bus bars 164 can bedisposed on opposing ends of transparent window 112. Electrical currentcan then be applied between bus bars 164 to heat the indium tin oxidecoating, which provides a consistent and uniform heat across transparentwindow 112 without interfering with fluorescence transmission. Bus bars164 can be controlled in response to control system 1010. In someembodiments, heating device 160 can be on both sides of transparentwindow 112.

Clamp Mechanism

In some embodiments, as seen in FIGS. 202-206, pressure chamber 150 canbe used with a clamp mechanism 1400 (best illustrated in FIGS. 204-206).Clamp mechanism 1400 can retain pressure chamber 150 in a clampedposition against thermocycler system 100.

Turning now to FIGS. 202 and 203, one of some embodiments of pressurechamber 150 is illustrated. A chamber body 1402 has a first side 1404and a second side 1406. In some embodiments, chamber body 1402 can beformed from aluminum or other materials such as steel, stainless steel,standard plastic, or fiber-reinforced plastic compound, such as a resinor polymer, and mixtures thereof. An opening 1408 extends through firstside 1404 and second side 1406.

A chamber cover 1410 has an opening 1412 surrounded by circumferentialchamber seal 154. Circumferential chamber seal 154 can have a peripherallip that 1413 that defines a sealing plane abutting sealing cover 80 ofmicroplate 20. In some embodiments, peripheral lip 1413 can bepositioned radially inward of a periphery of opening 1412. A reactivesurface 1415 can span between opening 1412 and peripheral lip 1413.Reactive surface 1415 can react to fluid pressure in pressure chamber150 by increasingly urging peripheral lip 1413 against sealing cover 80as the fluid pressure increases from zero to about 25 pounds per squareinch (PSI). In some embodiments, chamber cover 1410 is formed fromstainless steel. In some embodiments, a gasket 1414 (FIG. 203) can fitin a groove 1416 formed in a periphery of opening 1408 and provide aseal between chamber cover 1410 and chamber body 1402. Chamber cover1410 can be as thin as practicable and have a lower thermal mass thansaid chamber body to reduce heat flow between microplate 20 and chamberbody 1402. In some embodiments, frame 152 (also seen in FIG. 35) cancomprise chamber cover 1410 and chamber body 1402.

In some embodiments, a thin film heater 1418 can be positioned onchamber cover 1410 to further reduce heat flow into chamber body 1402.Thin film heater 1418 can have a heater signal input 1420 to receiveheater power from control system 1010. In some embodiments, athermocouple 1422 can be positioned on chamber cover 1410 and provide acover temperature signal 1424, by way of non-limiting example, via leadsor other signal transmission medium, to control system 1010.Thermocouple 1422 can comprise, by way of non-limiting example, a typeE, type J, type K, or type T thermocouple. Control system 1010 can usecover temperature signal 1424 to control heater power applied to thinfilm heater 1418 and thereby reduce temperature differences acrossmicroplate 20. In some embodiments, thin film heater 1418 can have apower dissipation of at least 50 watts.

In some embodiments, circumferential chamber seal 154 can be molded froma silicone material. In some embodiments, circumferential chamber seal154 can be insert-molded with chamber cover 1410. An alignment ring 1426can be fastened to chamber body 1402 through chamber cover 1410, andsecure chamber cover 1410 to second side 1406. Microplate 20 can fitwithin an inner periphery of alignment ring 1426. Alignment ring 1426can locate microplate 20 with respect to thermocycler system 100. Insome embodiments, an alignment feature 1428 can interface with alignmentfeature 58 of microplate 20. In some embodiments, recesses 1430 can beformed in the inner periphery of alignment ring 1426. Recesses 1430reduce a contact area between alignment ring 1426 and microplate 20 andcan thereby reduce heat flow between microplate 20 and alignment ring1426.

On first side 1404, a flange 1432 can protrude radially inward from theperiphery of opening 1408 and support a window seal 1434. In someembodiments, flange 1432 can be about ¼″ wide. A surface of transparentwindow 112 can abut window seal 1434. In some embodiments, for examplewhen window seal 1434 is a non-adhesive type seal, a window-retainingring 1436 can be secured to chamber body 1402 and clamp transparentwindow 112 against window seal 1434. A connector 1438 can provide aconnection to port 120 (FIGS. 34-37, 39-40) that is in fluidcommunication with the internal volume of pressure chamber 150.

At least one catch 1440 can be positioned on frame 152. In someembodiments, a pair of catches 1440 can be positioned on opposing sidesof a perimeter of frame 152. Each of the pair of catches 1440 can have acentering feature 1442.

Referring now to FIGS. 204-206, thermocycler system 100 and clampmechanism 1400 are illustrated fixedly mounted to a support structure1444. In some embodiments, support structure 1444 can be generallyplanar in construction and adapted to be mounted within housing 1008(FIG. 1). Clamp mechanism 1400 can be movable to between a lockedcondition (FIG. 204) and an unlocked condition (FIG. 205) and can beadapted to selectively clamp pressure chamber 150 against thermocyclersystem 100. An opening can be provided in support structure 1444 toallow contact between pressure chamber 150 and thermocycler system 100.In the locked condition, clamp mechanism 1400 can secure pressurechamber 150 in a clamped position against thermocycler system 100. Inthe clamped position, circumferential chamber seal 154 can be pressedagainst sealing cover 80 (best seen in FIG. 203). In the unlockedcondition, clamp mechanism 1400 can allow pressure chamber 150 to bemoved to an unclamped position away from thermocycler system 100. Insome embodiments, the unclamped position can provide a gap of ⅜ inchbetween thermocycler block 102 (FIG. 203) and microplate 20. In someembodiments, clamp mechanism 1400 can be actuated manually. In otherembodiments, clamp mechanism 1400 can be actuated by pneumatics,hydraulics, electric machines and/or motors, electromagnetics, or anyother suitable means.

In some embodiments, clamp mechanism 1400 can have a clamp frame 1446fixedly mounted to support structure 1444. An over-center link 1448 canpivot about a first end 1450 that can be pivotally connected to clampframe 1446. A bellcrank 1452 can pivot about a pivot pin 1454 connectedto clamp frame 1446. A lever arm 1456 can have a clamp end 1458pivotally connected to an input end 1460 of bellcrank 1452. Lever arm1456 can have an intermediate portion 1462 pivotally connected to asecond end 1464 of over-center link 1448. An input end 1466 of lever arm1456 can be pivotally connected to a telescoping end 1468 of a pneumaticcylinder 1470. A ball joint 1472 can pivotally connect telescoping end1468 to input end 1466. A mounting end 1474 of pneumatic cylinder 1470can pivotally connect to support structure 1444. In various otherembodiments, mounting end 1474 of pneumatic cylinder 1470 can pivotallyconnect to clamp frame 1446. Bellcrank 1452 can have a clamp end 1476. Aclamp pin 1478 can project from clamp end 1476 and engage centeringfeature 1442 when clamp mechanism 1400 is in the locked condition. Itshould be appreciated that the clamp mechanism 1400 on one side ofthermocycler system 100 has been described. A second clamp mechanism1401 can be positioned on the other side of thermocycler system 100(FIG. 206). Second clamp mechanism 1401 can be symmetrical with the sidejust described and operate similarly. A transverse member 1479 canconnect lever arm 1456 to the lever arm of the other side.

Operation of the clamp assembly 1400 embodiment illustrated in FIGS.204-206 will now be described. Pneumatic cylinder 1470 can be movablebetween an extended condition (FIG. 205) and a contracted condition(FIGS. 204 and 206). As pneumatic cylinder 1470 moves to the contractedcondition, it can cause lever arm 1456 to pivot as indicated by a curvedarrow A. Lever arm 1456 can in turn cause bellcrank 1452 to pivot asindicated by a curved arrow B, thereby moving clamp pin 1478 towardscentering feature 1442. Clamp pin 1478 can then become centered incentering feature 1442. As bellcrank 1452 completes rotating in thedirection of arrow B, it can cause clamp pin 1478 to move chamber 150from an unclamped position towards the clamped position againstthermocycler assembly 100. This can cause circumferential chamber seal154 to press against microplate 20 (best seen in FIG. 203). A clampingpressure between chamber seal 154 and microplate 20 can be adjusted byvarying the pivot location of first end 1450 of over-center link 1448.In some embodiments, an adjustment mechanism 1477, such as, by way ofnon-limiting example, a screw, can be used to vary the pivot location asindicated by arrows A (FIG. 205).

Moving clamp mechanism 1400 to the unlocked condition will now bedescribed. As pneumatic cylinder 1470 moves to the extended condition,it can cause lever arm 1456 to pivot in a direction opposite curvedarrow A. Lever arm 1456 can in turn cause bellcrank 1452 to pivot in adirection opposite curved arrow B, thereby relieving the clampingpressure between clamp pin 1478 and catch 1440. Clamp pin 1478 can thendisengage from centering feature 1442. As bellcrank 1452 completesrotating in the direction opposite curved arrow B, it can cause clamppin 1478 to move away from catch 1440, allowing chamber 150, withmicroplate 20, to move to the unclamped position away from thermocyclersystem 100.

In some embodiments, a pair of rails 1480 can be used to traversepressure chamber 150 between a thermocycler position adjacentthermocycler system 100 (FIG. 204) and a loading position away fromthermocycler system 100 (FIG. 205). In some embodiments, the loadingposition can be external of housing 1008. In such embodiments, housing1008 has an aperture that allows pressure chamber 150 and rails 1480 topass therethrough. In some embodiments, a position sensor 1487 can bepositioned on support structure 1440 and provide a position signalindicative of pressure chamber 150 being in the thermocycler position.In some embodiments, position sensor can be of an infrared, limitswitch, contactless proximity, or ultrasonic type. Rails 1480 can beslidably mounted to support structure 1444. In some embodiments, opticalsensor 1491 can read marking indicia 94 (FIG. 16) on microplate 20 as itis moved to the thermocycler position. Optical sensor 1491 can provide amarking data signal indicative of marking indicia 94 to control system1010.

In some embodiments, rails 1480 can be telescoping rails. Rails 1480 canbe moved manually or can be motorized. In some motorized embodiments, arack gear 1482 can be positioned on at least one of rails 1480. Arotating actuator 1484 can be adapted with a pinion gear 1486 thatengages rack gear 1482. Rotating actuator 1484 can rotate in response tocontrol signals from control system 1010. In some embodiments, rotatingactuator 1484 can be an electric motor, such as a stepper motor. Forexample, actuator 1484 can be a Vexta PK245-02AA stepper motor availablefrom Oriental Motor U.S.A. Corp. In other embodiments, rotating actuator1484 can be pneumatic or hydraulic. Pressure chamber 150 can be attachedbetween rails 1480.

In some embodiments, a lost motion mechanism 1488 can be positionedbetween rails 1480 and pressure chamber 150. Lost motion mechanism 1488can allow pressure chamber 150 limited perpendicular movement withrespect to rails 1480. The limited perpendicular movement facilitatesmoving pressure chamber 150 between the clamped and unclamped positionsas clamp assembly 1400 moves between the locked and unlocked conditions,respectively.

In some embodiments, lost motion mechanism 1488 can include shoulderbolts 1490 threaded into rails 1480. Catches 1440 can have through holes1492 that slidingly engage shoulder bolts 1490. In some embodiments,springs 1494 can be positioned between catches 1440 and rails 1480.Springs 1494 can bias pressure chamber 140 toward the unclamped positionand facilitate moving it away from thermocycler assembly 100 when clampassembly 1400 moves to the unlocked condition.

Pneumatic System

Referring now to FIGS. 207 and 208, a pneumatic system 1500 isillustrated in accordance with some embodiments. Pneumatic system 1500can provide pneumatic control for various pneumatic devices used insequence detection system 10. By way of non-limiting example, thepneumatic devices can include, alone or in any combination, pressurechamber 150, pneumatic cylinders 1470, and vacuum source 172.

An input coupling 1502 can provide a connection point for a supply ofcompressed fluid, such as, by way of non-limiting example, air, but canalso comprise nitrogen, argon, or helium. Input coupling 1502 can beaccessible from an exterior of housing 1008 (FIG. 1). In someembodiments, a pressure relief valve 1504 can be in fluid communicationwith input coupling 1502. In some embodiments, pressure relief valve1504 can have a maximum pressure of 120 PSI. In some embodiments, aparticle filter 1506 can be in fluid communication with pressure reliefvalve 1504. In some embodiments, a condensation separator 1508 can be influid communication with particle filter 1508. Alternatively,condensation separator 1508 can be in fluid communication with pressurerelief valve 1504. Particle filter 1506 and condensation separator 1508can provide a conditioned fluid supply 1510 to a remainder of pneumaticsystem 1500.

In some embodiments, a first pressure regulator 1512 can be in fluidcommunication with conditioned fluid supply 1510. First pressureregulator 1512 can provide a first fluid supply 1516 to a chamberpressurization subsystem 1518 and/or to other subsystems.

In chamber pressurization subsystem 1518, a check valve 1520 can beconnected in series with first pressure regulator 1512. Check valve 1520can reduce a risk of depressurization of the internal volume of pressurechamber 150 in the event conditioned fluid supply 1510 is interrupted. Aballast tank 1522 can be in fluid communication with the first fluidsupply 1516 and increase a fluid volume of chamber pressurizationsubsystem 1518. The increased volume can reduce pressure variations ofthe first fluid supply 1516. Ballast tank 1522 can also provide a fluidreserve to help maintain pressure in the event first fluid supply 1516is interrupted. One side of a charge valve 1524 can be in fluidcommunication with the first fluid supply 1516. The other side of chargevalve 1524 can be in fluid communication with the internal volume ofpressure chamber 150. A flexible fluid line can connect chamberpressurization subsystem 1518 to connector 1438 of chamber 150. Chargevalve 1524 can be controlled by control system 1010 in accordance with amethod described later herein. In some embodiments, charge valve 1524can be a part number MKH0NBG49A available from Parker-Hannifin Corp.

A pressure sensor 1526 can be in fluid communication with the internalvolume of pressure chamber 150 and can provide a chamber pressure signal1527 to control system 1010. In some embodiments, pressure sensor 1526can be a part number MPS-P6N-AG available from Parker-Hannifin Corp. Achamber pressure relief valve 1528 can be in fluid communication withthe internal volume of pressure chamber 150 and establish a maximumpressure that can be applied thereto. In some embodiments, the maximumpressure of 1528 chamber pressure relief valve can be less than, orequal to, 30 PSI.

Pressurization subsystem 1518 can also comprise a release valve 1530 influid communication with the internal volume of pressure chamber 150.The other side of release valve 1530 can be vented to atmosphere.Release valve 1530 can be controlled by control system 1010 inaccordance with a method described later herein. In some embodiments,release valve 1530 can be a part number MKH0NBG49A available fromParker-Hannifin Corp. In some embodiments, the charge and release valves1524, 1530 can maintain chamber pressure at about 18 PSI while themicroplate temperature is greater than 40 degrees Celsius. Thiscombination of pressure and temperature conditions can help reduce apossibility of pressure within wells 26 overcoming the chamber pressureand causing wells 26 to leak between sealing cover 80. A first silencer1532 can be in fluid communication with the other side of release valve1530 to reduce noise as fluid is vented.

In some embodiments, a second pressure regulator 1534 can be in fluidcommunication with conditioned fluid supply 1510. Second pressureregulator 1534 can provide a second fluid supply 1536 to a cylindercontrol subsystem 1538. Second pressure regulator 1540 can also providesecond fluid supply 1536 to a vacuum control subsystem 1540. A pressuretransducer 1542 can be in fluid communication with second fluid supply1536 and provide a pressure signal 1544 to control system 1010. In someembodiments, pressure transducer 1542 can comprise a part numberMPS-P6N-AG available from Parker-Hannifin Corp. In some embodiments,second fluid supply 1536 is greater than, or equal to, 50 PSI.

In cylinder control subsystem 1538, a cylinder valve 1546 can have apressure port 1548, an exhaust port 1550, a first port 1552, and asecond port 1554. Cylinder valve 1546 can be referred to as a3-position, 2-port valve, commonly referred to as a 3/2 valve. In someembodiments, cylinder valve 1546 can comprise a part numberP2MISGEE2CV2DF7 available from Parker-Hannifin Corp. or a part numberB360BA549C available from Parker-Hannifin Corp. Pressure port 1548 canbe in fluid communication with second fluid supply 1536. Exhaust port1550 can be vented to atmosphere. Cylinder silencer 1556 can be in fluidcommunication with exhaust port 1550 to reduce noise when fluid isvented from pneumatic cylinder 1470. First port 1552 can be in fluidcommunication with first port 1558 of pneumatic cylinder 1470. Secondport 1554 can be in fluid communication with second port 1559 ofpneumatic cylinder 1470. Cylinder valve 1546 can be manually controlled.In some embodiments, cylinder valve 1546 is a servovalve controlled bycontrol system 1010 in accordance with a method described later herein.

Cylinder valve 1546 can have three positions that route fluid betweenports 1548-1554. A first position can route pressure port 1548 to firstport 1552 and route second port 1554 to exhaust port 1550. A secondposition can block pressure port 1548 and route first and second ports1552, 1554 to exhaust port 1550. A third position can route pressureport 1548 to second port 1554 and route first port 1552 to exhaust port1550. The first, second, and third positions of cylinder valve 1546 canbe referred to as the lock, release, and unlock positions, respectively.

When cylinder valve 1546 is in the lock position, fluid routing throughcylinder valve 1546 can cause pneumatic cylinder 1470 to move to thecontracted condition, thereby moving clamp mechanism 1400 to the lockedcondition (FIG. 204). When cylinder valve 1546 is in the unlockposition, the fluid routing through cylinder valve 1546 can causepneumatic cylinder 1470 to move to the extended condition, therebymoving clamp mechanism 1400 to the unlocked condition (FIG. 205). Whencylinder valve 1546 is in the release position, the fluid routingthrough cylinder valve 1546 can cause pneumatic cylinder 1470 to befreely extended or contracted by an outside influence, thereby allowingclamp mechanism 1400 to be manually moved between the closed and openpositions. It should be noted that over-center link 1448 can maintainclamp mechanism in the locked condition when cylinder valve 1546 ismoved to the release position. A first limit switch 1560 can sense,either directly or indirectly, when pneumatic cylinder 1470 is in theextended condition and provide a corresponding signal 1562 to controlsystem 1010. A second limit switch 1564 can be used to sense, eitherdirectly or indirectly, when pneumatic cylinder 1470 is in thecontracted condition and provide a corresponding signal 1566 to controlsystem 1010. In some embodiments, first and second limits switches 1560,1564 can be integral to pneumatic cylinder 1470. In some embodiments,pneumatic cylinder 1470 can be a Parker-Hannifin Corp. SRM Seriespneumatic cylinder with piston sensing capability. In some embodiments,pneumatic cylinder 1470 can be a part number L06DP-SRMBSY400 fromParker-Hannifin Corp.

In some embodiments, vacuum control system 1540 selectively actuatesvacuum source 172. Vacuum generated by vacuum source 172 can be providedto thermocycler system 100 or other systems. Vacuum control system 1572can comprise a vacuum control valve 1568. In some embodiments, vacuumcontrol valve 1568 can comprise a part number P2MISDEE2CV2BF7 availablefrom Parker-Hannifin Corp.

Vacuum control valve 1568 can have a pressure port 1570, an exhaust port1572, a first port 1574, and a second port 1576. Vacuum control valve1568 can be referred to as a 3-position, 2-port valve, commonly referredto as a 3/2 valve. Pressure port 1570 can be in fluid communication withsecond fluid supply 1536. In some embodiments, exhaust port 1572 can beblocked. In other embodiments, exhaust port 1572 can be vented toatmosphere. First port 1574 can be in fluid communication with vacuumsource 172. Second port 1576 can be blocked in some embodiments havingexhaust port 1572 vented to atmosphere. In other embodiments, secondport 1576 can be vented to atmosphere. Vacuum control valve 1568 can bemanually controlled. In some embodiments, vacuum control valve 1568 is aservovalve controlled by control system 1010 in accordance with a methoddescribed later herein.

Vacuum control valve 1568 can have three positions that route fluidbetween ports 1570-1576. A first position can route pressure port 1570to first port 1574, and can block exhaust port 1572 and second port1576. A second position can block pressure port 1570, and route firstand second ports 1574, 1576 through exhaust port 1572. A third positioncan route pressure port 1570 to second port 1576, and block first port1574 and exhaust port 1572. The first, second, and third positions ofvacuum control valve 1568 can also be referred to as the vacuum on,vacuum off, and vent positions, respectively.

When vacuum control valve 1568 is in the vacuum on position, the fluidrouting through vacuum control valve 1568 can flow through vacuum source172. Vacuum source 172 generates a vacuum in response thereto that canbe fluidly coupled to the thermocycler system 100 or other systems. Whenvacuum control valve 1568 is in the vacuum off position, second fluidsupply 1536 is disconnected from vacuum source 172 and vacuum source 172can be routed to atmosphere through exhaust port 1572 and/or second port1576. When vacuum control valve 1568 is in the vent position, secondfluid supply 1536 can be purged to atmosphere through second port 1576.

Referring now to FIG. 209, a method 1580 is illustrated, according tosome embodiments, for clamping pressure chamber 150 to thermocyclersystem 100. Method 1580 can be executed by control system 1010 whenpressure chamber 150 is placed in proximity to thermocycler block 102.Method 1580 can begin in step 1582 and can proceed to decision step 1584to determine whether pressure chamber 150 is properly located withinclamp mechanism 1400. Position signal 1489 (FIG. 204) can be used tomake the determination. When pressure chamber 150 is properly located,method 1580 can proceed to step 1586 and move cylinder valve 1546 to thelock position. Method 1580 can then proceed to decision step 1588 anddetermine whether pneumatic cylinder 1470 has moved to the contractedcondition, thereby placing clamp mechanism 1400 in the locked condition.Decision step 1588 can make the determination by using signal 1566 (FIG.207) from second limit switch 1570. Method 1580 can execute decisionstep 1588 until pneumatic cylinder 1470 moves to the contractedcondition. Method 1580 can then proceed to step 1590 and can perform aleak test 1590 as described later herein. Method 1580 can then proceedto decision step 1592 and determine, from results of leak test 1590,whether leak test 1590 passed. If leak test 1590 passed, then method1580 can proceed to step 1594 and exit. If leak test 1590 failed, thenmethod 1580 can proceed to step 1610 and release chamber 150 accordingto a method described later herein.

Returning to decision step 1584, if method 1580 determines that chamber150 is improperly located within clamp mechanism 1400, then method 1580can proceed to step 1596. In step 1596, method 1580 can indicate thatchamber 150 is improperly located within clamp mechanism 1400. Method1580 can then proceed to method 1610 and assure clamp mechanism 1400 isin the unlocked condition. Method 1580 can indicate the improperlocation of chamber 150 though, by way of example, a buzzer, lamp,writing to a computer memory in control system 1010, or any othersuitable means.

Referring now to FIG. 210, method 1590 is illustrated, according to someembodiments of the invention, for performing the leak test on chamber150. Method 1590 can be executed by control system 1010 when chamber 150is in the clamped position. Method 1590 can begin at step 1591 and canproceed to step 1593. In step 1593, method 1590 can pressurize chamber150 by opening charge valve 1524 and closing release valve 1530 (FIG.207). Method 1590 can then proceed to decision step 1595 and determine achamber leak rate of pressure chamber 150. In one of some embodiments,the chamber leak rate can be determined by determining a difference inair pressure, as indicated by pressure transducer 1526, over apredetermined amount of time. In one example, the chamber leak rate canbe expressed in units of PSI/minute. In decision step 1595, method 1590can compare the chamber leak rate to a predetermined leak rate. If thechamber leak rate is less than the predetermined leak rate, method 1590can proceed to step 1598, indicating that the leak test has passed.Method 1590 can then proceed to step 1600 and open charge valve 1524 toconnect ballast tank 1536 to the internal volume of pressure chamber150. In step 1600, method 1590 can also provide an indication to controlsystem 1010 that thermocycling can begin.

Returning now to decision step 1595, if the chamber leak rate is greaterthan, or equal to, the predetermined leak rate, method 1590 can proceedto step 1602, indicating that the leak test has failed. Method 1590 canthen proceed to step 1604 and indicate the failure though, by way ofexample, a buzzer, lamp, writing to the computer memory in controlsystem 1010, or any other suitable means. Method 1590 can exit at step1606 from either step 1600 or step 1604.

Referring now to FIG. 211, method 1610 of unclamping pressure chamber150 from thermocycler system 100 is illustrated according to one ofseveral embodiments. Method 1610 can be executed by control system 1010.In some embodiments, method 1612 can be called by method 1580. Method1610 can also be executed after thermocycling is completed. Method 1610can begin in step 1612 and then can proceed to step 1614. In step 1614,method 1610 can move cylinder valve 1546 to the unlock position, whichcan cause pneumatic cylinder 1470 to begin moving to the extendedcondition and changing clamp mechanism to the unlocked condition. Method1610 can then proceed to decision step 1616 and determine whetherpneumatic cylinder 1470 has moved to the extended condition. Decisionstep 1616 can make the determination by using signal 1562 (FIG. 207)from first limit switch 1560. Method 1610 can execute decision step 1616until pneumatic cylinder 1470 moves to the extended condition. Method1610 can then proceed to step 1618 and exit.

Excitation System

In some embodiments, as illustrated in FIGS. 42-49, excitation system200 generally comprises a plurality of excitation lamps 210 generatingexcitation light 202 in response to control signals from control system1010. Excitation system 200 can direct excitation light 202 to each ofthe plurality of wells 26 or across the plurality of wells 26. In someembodiments, excitation light 202 can be a radiant energy comprising awavelength that permits detection of photo-emitting detection probes inassay 1000 disposed in at least some of the plurality of wells 26 ofmicroplate 20 by detection system 300.

By way of background, it should be understood that the quantitativeanalysis of assay 1000, in some embodiments, can involve measurement ofthe resultant fluorescence intensity or other emission intensity. Insome embodiments of the present teachings, fluorescence from theplurality of wells 26 on microplate 20 can be measured simultaneouslyusing a CCD camera. In an idealized optical system, if all of theplurality of wells 26 have the same concentration of dye, each of theplurality of wells 26 would produce an identical fluorescence signal. Insome prior conventional designs, wells near the center of the microplatemay appear significantly brighter (i.e. output more signal) than thosewells near the edge of the microplate, despite the fact that all of thewells may be outputting the same amount of fluorescence. There areseveral reasons for this condition in some current designs—vignetting,shadowing, and the particular illumination/irradiance profile.

With respect to vignetting, camera lenses can collect more light fromthe center of the frame relative to the edges. This can reduce theefficiency of certain prior, conventional detection systems.Additionally, in certain prior, conventional designs, the irradianceprofile is sometimes not uniform. Most commercially available irradiancesources have a greater irradiance value (watts/meter²) near the centercompared to the edges of the irradiance zone. In PCR, it has been foundthat for a given dye, until the dye saturates or bleaches, the amount offluorescence can be proportional to the irradiance of the illuminationsource. Therefore, if the excitation light is brighter at the center,then the fluorescence signal from a well near the edge of the irradiancezone would be less than an identical well near the center. Shadowing canoccur due to the depth of the wells. Unless the excitation light isperpendicular to the microplate, some part of the well may not beproperly illuminated. In other words, the geometry of the well may blocksome of the light from reaching the bottom of the well. In addition, theamount of fluorescence emitted, which can be collected, may vary fromcenter to edge. As should be appreciated by one skilled in the art,noise sources are often constant across the field of view of the camera.Therefore, for wells near the edges of microplate 20 that output asmaller amount of fluorescence, the signal to noise ratio can beadversely effected, thereby reducing the efficiency of high-densitysequence detection system 10. As illustrated in FIG. 50, a graphillustrates the relative intensity or light transmission versus welllocation on a plate. As can be seen from the graph, the effects ofvignetting and shadowing causes the light intensity to drop off alongthe edges of the field of view of the plate.

The present teachings, at least in part, address these effects so thatidentical wells output generally identical fluorescence irrespective oftheir location on microplate 20. By using the profile from FIG. 50, theoptimum irradiance profile can be calculated. With reference to FIG. 51,a corresponding irradiance profile, represented by a dashed line, canprovide a higher irradiance along the edges. This irradiance profile,when coupled with the effects of vignetting and shadowing, createsgenerally uniform signal strength across all of the plurality of wells26 of microplate 20.

Excitation Sources

In some embodiments, as illustrated in FIGS. 42-49, the plurality ofexcitation lamps 210 of excitation system 200 can be fixedly mounted toa support structure 212. In some embodiments, the plurality ofexcitation lamps 210 can be removably mounted to support structure 212to permit convenient interchange, exchange, replacement, substitution,or the like. In some embodiments, support structure 212 can be generallyplanar in construction and can be adapted to be mounted within housing1008 (FIG. 1). The plurality of excitation lamps 210 can be arranged ina generally circular configuration and directed toward microplate 20 topromote uniform excitation of assay 1000 in each of the plurality ofwells 26. The present teachings permit a generally uniform excitationthat is substantially free of shadowing. In some embodiments, theplurality of excitation lamps 210 can be arranged in a generallycircular configuration about an aperture 214 formed in support structure212. Aperture 214 permits the free transmission of fluorescencetherethrough for detection by detection system 300, as described herein.

In some embodiments, as illustrated in FIGS. 52-56, each of theplurality of excitation lamps 210 can be configured to achieve thedesired irradiance profile. In some embodiments, as seen schematicallyin FIG. 52, each of the plurality of excitation lamps 210 can comprise alens 216. Lens 216 can be shaped to provide a desired irradiance profile(see FIG. 51). The exact shape of lens 216 can depend, at least in part,upon one or more of the desired irradiance profile at microplate 20, theillumination/irradiance profile at each of the plurality of excitationlamps 210, and the size and position of microplate 20 relative to theplurality of excitation lamps 210. The shape of lens 216 can becalculated in response to the particular application using commerciallyavailable software, such as ZEMAX and/or ASAP.

In some embodiments, as seen schematically in FIG. 53, each of theplurality of excitation lamps 210 can comprise a mirror 218. Mirror 218can be shaped to provide a desired irradiance profile (see FIG. 51). Theexact shape of mirror 218 can be dependent, at least in part, upon thedesired irradiance profile at microplate 20, the illumination/irradianceprofile at each of the plurality of excitation lamps 210, and the sizeand position of microplate 20 relative to the plurality of excitationlamps 210. The shape of mirror 218 can be calculated in response to theparticular application using commercially available software, such asZEMAX and/or ASAP.

In some embodiments, as illustrated in FIG. 54, each of the plurality ofexcitation lamps 210 can comprise a combination of lens 216 and mirror218 to achieve the desired irradiance profile. Again, lens 216 andmirror 218 can be calculated in response to the particular applicationusing commercially available software, such as ZEMAX and/or ASAP.

Turning now to FIG. 55, in some embodiments, each of the plurality ofexcitation lamps 210 can be aligned such that their optical centersconverge on a single point 220. Additionally, in some embodiments, adesired irradiance profile (see FIG. 51) can be achieved by directingeach of the plurality of excitation lamps 210 at a predeterminedlocation 222 a-222 n on microplate 20, as illustrated in FIG. 56. Insome embodiments, each of the plurality of excitation lamps 210 cancomprise lens 216 and/or mirror 218 and can further be aligned asillustrated in FIG. 56 to achieve more complex irradiance profiles. Ascan be appreciated, employing any of the above techniques describedherein can provide improved irradiance across microplate 20, therebyimproving the resultant signal to noise ratio of the plurality of wells26 along the edge of microplate 20.

It is anticipated that the plurality of excitation lamps 210 can be anyone of a number of sources. In some embodiments, the plurality ofexcitation lamps 210 can be a laser source having a wavelength of about488 nm, an Argon ion laser, an LED, a halogen bulb, or any other knownsource. In some embodiments, the LED can be a MR16 from OptoTechnologies (Wheeling Ill.; http://www.optotech.com/MR16.htm). In someembodiments, the LED can be provided by LumiLEDS. In some embodiments,the halogen bulb can be a 75 W, 21 V DC lamp or a 50 W, 12 V DC lamp.

As discussed above, each of the plurality of excitation sources 210 canbe removably coupled to support structure 212 to permit convenientinterchange, exchange, replacement, substitution, or the like thereof.In some embodiments, the particular excitation source(s) employed can beselected by one skilled in the art to exhibit desired characteristics,such as increased power, better efficiency, improved uniformity,multi-colors, or having any other desired performance criteria. Inembodiments employing multi-color and/or multi-wavelength excitationsources, additional detection probes and/or dyes can be used to, in somecircumstances, increase throughput of high-density sequence detectionsystem 10 by including multiple assays in each of the plurality of wells26.

In some embodiments, the temperature of the plurality of excitationlamps 210 can be controlled to decrease the likelihood of intensity andspectral shifts. In such embodiments, the temperature control can be,for example, a cooling device. In some embodiments, the temperaturecontrol can maintain each of the plurality of excitation lamps 210 at anessentially constant temperature. In some embodiments, the intensity canbe controlled via a photodiode feedback system, utilizing pulse widthmodulation (PWM) control to modulate the power of the plurality ofexcitation lamps 210. In some embodiments, the PWM can be digital. Insome embodiments, shutters can be used to control each of the pluralityof excitation lamps 210. It should be appreciated that any of theexcitation assemblies 200 illustrated in FIGS. 42-49 and described abovecan be interchanged with each other.

Detection Systems

In some embodiments, as illustrated in FIGS. 42-44, 47, and 48,detection system 300 can be used to detect and/or gather fluorescenceemitted from assay 1000 during analysis. In some embodiments, detectionsystem 300 can comprise a collection mirror 310, a filter assembly 312,and a collection camera 314. After excitation light 202 passes into eachof the plurality of wells 26 of microplate 20, assay 1000 in each of theplurality of wells 26 can be illuminated, thereby exciting a detectionprobe disposed therein and generating an emission (i.e. fluorescence)that can be detected by detection system 300.

In some embodiments, collection mirror 310 can collect the emissionand/or direct the emission from each of the plurality of wells 26towards collection camera 314. In some embodiments, collection mirror310 can be a 120 mm-diameter mirror having ¼ or ½ wave flatness and40/20 scratch dig surface. In some embodiments, filter assembly 312comprises a plurality of filters 318. During analysis, microplate 20 canbe scanned numerous times—each time with a different filter 318.

In some embodiments, collection camera 314 comprises a multi-elementphoto detector 324, such as, but not limited to, charge coupled devices(CODs), diode arrays, photomultiplier tube arrays, charge injectiondevices (CIDs), CMOS detectors, and avalanche photodiodes. In someembodiments, the emission from each of the plurality of wells 26 can befocused on collection camera 314 by a lens 316. In some embodiments,collection camera 314 is an ORCA-ER cooled CCD type available fromHamamatsu Photonics. In some embodiments, lens 316 can have a focallength of 50 mm and an aperture of 2.0. In some embodiments, collectioncamera 314 can be mounted to, and prealigned with, lens 316.

In some embodiments, detection system 300 can comprise a lightseparating element, such as a light dispersing element. Light dispersingelement can comprise elements that separate light into its spectralcomponents, such as transmission gratings, reflective gratings, prisms,beam splitters, dichroic filters, and combinations thereof that are canbe used to analyze a single bandpass wavelength without spectrallydispersing the incoming light. In some embodiments, with a singlebandpass wavelength light dispersing element, a detection system can belimited to analyzing a single bandpass wavelength. Therefore, one ormore light detectors, each comprising a single bandpass wavelength lightdispersing element, can be provided.

In some embodiments, as seen in FIG. 212, an alignment mount 320 canmate collection camera 314 and lens 316. Alignment mount 320 can providea mechanism to adjust an axial alignment and a distance between an opticassembly 322 and multi-element photo detector 324. Lens 316 can receiveoptic assembly 322 and can mount to a mounting face 326 of a base plate328. Base plate 328 can have an aperture 330 formed therein that canallow light to pass from optic assembly 322 to multi-element photodetector 324. In some embodiments, base plate 328 can be formed from ametal, such as steel, stainless steel, or aluminum.

Collection camera 314 can contain multi-element photo detector 324 andcan mount to a camera mounting plate 332. Mounting plate 332 can have anaperture 334 that can align with aperture 330. Mounting plate 332 canhave a face 336 generally parallel to a mating face 338 of base plate328. In some embodiments, mounting plate 332 can be formed from a metal,such as steel, stainless steel, or aluminum. At least one resilientmember 340 can attach to mounting plate 332 and to base plate 328.Resilient member 340 can be formed, by non-limiting example, from aspring and/or other elastic structure. Resilient member 340 can providea bias force that urges face 336 towards mating face 338. A planarityadjustment feature, such as, by way of non-limiting example, at leastone setscrew 342, can be positioned between face 336 and mating face338. At least one setscrew 342 can apply a force opposite the bias forceprovided by resilient member 340 and maintain face 336 in a spacedrelationship from mating face 338.

In some embodiments, at least one set screw 342 can have a thread pitchbetween 80 and 100 threads per inch (TPI), inclusive. In someembodiments, at least one setscrew 342 can be a ball-end type. In someembodiments, three setscrews 342 can be radially spaced around mountingplate 332. In some embodiments, the planarity adjustment feature cancomprise cams, motorized screws, fluid-containing bags, or inclinedplanes. In some embodiments, the space between face 336 and mating face338 can be less than ⅛ inch. In some embodiments, a light blockinggasket 344 can be positioned in the space between face 336 and matingface 338. In some embodiments, light blocking gasket 344 can be formedfrom closed cell foam. Light blocking gasket 344 can have aperturesformed therein that align with apertures 330 and 334, and with theplanarity adjustment feature.

In some embodiments, at least one of collection camera 314 and lens 316can have a mount comprising a threaded mount or a bayonet mount. Thethreaded mount can comprise, for example, a C-mount or a CS-mount. Thebayonet mount can comprise, for example, an F-mount or a K-mount. Insome embodiments, collection camera 314 can be mounted to mounting plate332 using a mounting ring 346 and a retaining ring 348. In someembodiments, mounting plate 332 can be formed from a metal, such assteel, stainless steel, or aluminum. Collection camera 314 can besecured to mounting ring 346. Mounting ring 346 can fit into a groove350 formed around a periphery of aperture 334. Retaining ring 348 canfasten to mounting plate 332 and can cover at least a portion of groove350 and a portion of mounting ring 346, thereby retaining mounting ring346 within groove 350. In some embodiments, retaining ring 348 can beformed from a metal, such as steel, stainless steel, or aluminum. Insome embodiments, a concentricity adjustment feature, such as at leastone set screw 352, can protrude radially into groove 350 and can pressagainst an outer periphery 354 of mounting ring 346. The concentricityadjustment feature can locate mounting ring 350 in an x-y plane ofgroove 350. The x-y plane can be illustrated by a coordinate system 356.In some embodiments, at least one setscrew 352 can have a thread pitchbetween 80 TPI and 100 TPI, inclusive. In some embodiments, at least onesetscrew 352 can be a ball-end type. The concentricity adjustmentfeature in other embodiments can include cams, motorized screws,fluid-containing bags, and/ or inclined planes.

A line segment 358 can represent an image plane of optic assembly 322.An arrow 360 can be centered on optic assembly 322 and normal to itsimage plane 358. A line segment 362 can represent an image plane ofmulti-element photo detector 324. An arrow 364 can be centered onmulti-element photo detector 324 and normal to its image plane 362.

In operation, the planarity adjustment feature, such as at least one setscrew 342, can be used to tilt mounting plate 332 such that image plane362 can become parallel with image plane 322. The planarity adjustmentfeature can also used to adjust the distance between optic assembly 322and multi-element photo detector 324.

The concentricity adjustment feature, such as at least one setscrew 352,can translate mounting ring 346 in the x-y plane. Translating mountingring 346 can adjust arrow 364 concentrically with arrow 360.

In some embodiments, alignment features 368 can align base plate 328with support structure 212. Locations of alignment features 368 anddimensions of alignment mount 320 can be selected to place the arrow 360concentric with a center of microplate 20. Locations of alignmentfeatures 356 and dimensions of alignment mount 320 can be selected toplace image plane 358 in parallel with an image plane of microplate 20.In some embodiments having collection mirror 310 (of FIGS. 42 and 43),locations of alignment features 356 and dimensions of alignment mount320 can be selected to place image plane 358 perpendicular with theimage plane of microplate 20. In some embodiments, base plate 328 caninclude a foot plate 366. By way of non-limiting example, alignmentfeatures 368 can comprise any combination of dowels and keys.

Control System

In some embodiments, control system 1010 can be operable to controlvarious portions of high-density sequence detection system 10 and tocollect data. In such embodiments, control system 1010 can comprisesoftware and devices operable to collect and analysis data; controloperation of electrical, mechanical, and optical portions ofhigh-density sequence detection system 10; and thermocycling. In someembodiments, such data analysis can comprise organizing, manipulating,and reporting of data and derived results to determine relative geneexpression within assay 1000, between various test samples, and acrossmultiple test runs.

In some embodiments, control system 1010 can archive data within adatabase, database retrieval, database analysis and manipulation, andbioinformatics. In some embodiments, control system 1010 can be operableto analyze raw data and among other actions, control operation ofhigh-density sequence detection system 10. Such analysis of raw data cancomprise compensating for point spread (PSF), background or baseemissions, a unique intensity profile, optical crosstalk, detectorand/or optical path variability and noise, misalignment, or movementduring operation. This can be accomplished, in some embodiments, byutilizing internal controls in several of the plurality of wells 26, aswell as calibrating high-density sequence detection system 10. In someembodiments, data analysis can comprise difference imaging, such ascomparing an image from one point in time to an image at a differentpoint in time, or image subtracting. In some embodiments, data analysiscan comprise curve fitting based on a specific gene or a gene set. Stillfurther, in some embodiments, data analysis can comprise using notemplate control (NTC) background or baseline correction. In someembodiments, data analysis can comprise error estimation usingconfidence values derived in terms of CT. See U.S. Patent ApplicationNo. 60/517,506 filed Nov. 4, 2003 and U.S. Patent Application No.60/519,077 filed Nov. 10, 2003.

In some embodiments, the present teachings can provide a method forreducing signal noise from an array of pixels of a segmented detectorfor biological samples. The signal noise comprises a dark currentcontribution and readout offset contribution. The method can compriseproviding a substantially dark condition for the array of pixels,wherein the dark condition comprises being substantially free offluorescent light emitted from the biological samples, providing a firstoutput signal from a binned portion of the array of pixels by collectingcharge for a first exposure duration, transferring the collected chargeto an output register and reading out the register, wherein transferringof the collected charge from the binned pixels comprises providing agate voltage to a region near the binned pixels to move collected chargefrom the binned pixels, and wherein the collected charge can betransferred in a manner that causes the collected charge to be shiftedto the output register, providing a second output signal from each pixelby collecting charge for a second exposure duration, transferring thecollected charge to the output register, and reading out the register,providing a third output signal by resetting and reading out the outputregister, determining the dark current contribution and the readoutoffset contribution from the first output signal, the second outputsignal, and the third output signal.

In some embodiments, the present teachings can provide a method ofcharacterizing signal noise associated with operation of acharge-coupled device (CCD) adapted for analysis of biological samples,wherein the signal noise comprises a dark current contribution, readoutoffset contribution, and spurious change contribution. The method cancomprise providing a plurality of first data points associated withfirst outputs provided from the CCD under a substantially dark conditionduring a first exposure duration, providing a plurality of second datapoints associated with second outputs provided from the CCD under thesubstantially dark condition during a second exposure duration whereinthe second duration is different from the first duration, providing aplurality of third data points associated with third outputs providedfrom a cleared output register of the CCD without comprising chargetransferred thereto, determining the dark current contribution per unitexposure time by comparing the first data points and the second datapoints, determining the readout offset contribution from the third datapoints, and determining the spurious charge contribution based on thedark current contribution and the readout offset contribution. See U.S.patent application Ser. No. 10/913,601 filed Aug. 5, 2004; U.S. patentapplication Ser. No. 10/660,460 filed Sep. 11, 2003, and U.S. patentapplication Ser. No. 10/660,110 filed Sep. 11, 2003.

Methods of Use and Analysis

Polynucleotide Amplification

In some embodiments, a high-density sequence detection system orcomponents thereof are used for the amplification of polynucleic acids,such as by PCR. Briefly, by way of background, PCR can be used toamplify a sample of target Deoxyribose Nucleic Acid (DNA) for analysis.Typically, the PCR reaction involves copying the strands of the targetDNA and then using the copies to generate additional copies insubsequent cycles. Each cycle doubles the amount of the target DNApresent, thereby resulting in a geometric progression in the number ofcopies of the target DNA. The temperature of a double-stranded targetDNA is elevated to denature the DNA, and the temperature is then reducedto anneal at least one primer to each strand of the denatured targetDNA. In some embodiments, the target DNA can be a cDNA. In someembodiments, primers are used as a pair—a forward primer and a reverseprimer—and can be referred to as a primer pair or primer set. In someembodiments, the primer set comprises a 5′ upstream primer that can bindwith the 5′ end of one strand of the denatured target DNA and a 3′downstream primer that can bind with the 3′ end of the other strand ofthe denatured target DNA. Once a given primer binds to the strand of thedenatured target DNA, the primer can be extended by the action of apolymerase. In some embodiments, the polymerase can be a thermostableDNA polymerase, for example, a Taq polymerase. The product of thisextension, which sometimes may be referred to as an amplicon, can thenbe denatured from the resultant strands and the process can be repeated.Temperatures suitable for carrying out the reactions are well known inthe art. Certain basic principles of PCR are set forth in U.S. Pat. Nos.4,683,195, 4,683,202, 4,800,159, and 4,965,188, each issued to Mullis etal.

In some embodiments, PCR can be conducted under conditions allowing forquantitative and/or qualitative analysis of one or more target DNA.Accordingly, detection probes can be used for detecting the presence ofthe target DNA in an assay. In some embodiments, the detection probescan comprise physical (e.g., fluorescent) or chemical properties thatchange upon binding of the detection probe to the target DNA. Someembodiments of the present teaching can provide real timefluorescence-based detection and analysis of amplicons as described, forexample, in PCT Publication No. WO 95/30139 and U.S. patent applicationSer. No. 08/235,411.

In some embodiments, assay 1000 can be a homogenous polynucleotideamplification assay, for coupled amplification and detection, whereinthe process of amplification generates a detectable signal and the needfor subsequent sample handling and manipulation to detect the amplifiedproduct is minimized or eliminated. Homogeneous assays can provide foramplification that is detectable without opening a sealed well orfurther processing steps once amplification is initiated. Suchhomogeneous assays 1000 can be suitable for use in conjunction withdetection probes. For example, in some embodiments, the use of anoligonucleotide detection probe, specific for detecting a particulartarget DNA can be included in an amplification reaction in addition to aDNA binding agent of the present teachings. Homogenous assays amongthose useful herein are described, for example, in commonly assignedU.S. Pat. No. 6,814,934.

In some embodiments, methods are provided for detecting a plurality oftargets. Such methods include those comprising forming an initialmixture comprising an analyte sample suspected of comprising theplurality of targets, a polymerase, and a plurality of primer sets. Insome embodiments, each primer set comprises a forward primer and areverse primer and at least one detection probe unique for one of theplurality of primer sets. In some embodiments, the initial mixture canbe formed under conditions in which one primer elongates if hybridizedto a target.

In some embodiments, the location of a fluorescent signal on a solidsupport, such as microplate 20, can be indicative of the identity of atarget comprised by the analyte sample. In some embodiments, a pluralityof detection probes are distributed to identify loci of at least some ofthe plurality of wells 26 of microplate 20. A signal deriving from adetection probe, such as, for example, an increase in fluorescenceintensity of a fluorophore at a particular locus can be detected if anamplification product binds to a detection probe and is then amplified.The location of the locus can indicate the identity of the target, andthe intensity of the fluorescence can indicate the quantity of thetarget.

In some embodiments, reagents are provided comprising a master mixcomprising at least one of catalysts, initiators, promoters, cofactors,enzymes, salts, buffering agents, chelating agents, and combinationsthereof. In some embodiments, reagents can include water, a magnesiumcatalyst (such as MgCl2), polymerase, a buffer, and/or dNTP. In someembodiments, specific master mixes can comprise AmpliTaq® Gold PCRMaster Mix, TaqMan® Universal Master Mix, TaqMan® Universal Master MixNo AmpErase® UNG, Assays-by-Design^(SM), Pre-Developed Assay Reagents(PDAR) for gene expression, PDAR for allelic discrimination andAssays-On-Demand®, (all of which are marketed by Applied Biosystems).However, the present teachings should not be regarded as being limitedto the particular chemistries and/or detection methodologies recitedherein, but may employ Taqman®; Invader®; Taqman Gold®; protein,peptide, and immuno assays; receptor binding; enzyme detection; andother screening and analytical methodologies.

In some embodiments, high-density sequence detection system 10 isoperable for analysis of materials (e.g., polynucleotides) comprising orderived from genetic materials from organisms. In some embodiments, suchmaterials comprise or are derived from substantially the entire genomeof an organism. In some embodiments, such organisms include, forexample, humans, mammals, mice, Arabidopsis or any other plant,bacteria, fungi, or animal species. In some embodiments, assay 1000comprises at least one of a homogenous solution of a DNA sample, atleast one primer set for detection of a polynucleotide comprising orderived from such genetic materials, at least one detection probe, apolymerase, and a buffer. In some embodiments, assay 1000 comprises atleast one of a plurality of different detection probes and/or primersets to perform multiplex PCR, which can be particularly useful whenanalyzing a whole genome having, for example, about 30,000 differentgenes. In some embodiments, analysis of substantially the entire genomeof an organism is conducted on a single microplate 20, or on multiplemicroplates (e.g., two, three, four or more) each comprising subparts ofsuch materials comprising or derived from the genetic materials of theorganism. In some embodiments using multiple microplates, a plurality ofplates contain a plurality of assay 1000 having essentially identicalmaterials and a plurality of assay 1000 having different materials. Insome embodiments, a plurality of plates do not contain assay 1000 havingessentially identical materials. In some embodiments, microplate 20comprises a fixed subset of a genome. It should also be recognized thatthe present teachings can be used in connection with genotyping, geneexpression, or other analysis.

In various some embodiments, the microplate can be covered with asealing liquid prior to performance of analysis or reaction of assay1000. For example, in some embodiments, a sealing liquid is applied tothe surface of a microplate comprising reaction spots comprising anassay 1000 for amplification of polynucleotides. In some embodiments, asealing liquid can be a material which substantially covers the materialretention regions (e.g., reaction spots) on the microplate so as tocontain materials present in the material retention regions, andsubstantially prevent movement of material from one reaction region toanother reaction region on the substrate. In some embodiments, thesealing liquid can be any material which is not reactive with assay 1000under normal storage or usage conditions. In some embodiments, thesealing liquid can be substantially immiscible with assay 1000. In someembodiments, the sealing liquid can be transparent, have a refractiveindex similar to glass, have low or no fluorescence, have a lowviscosity, and/or be curable. In some embodiments the sealing liquid cancomprise a flowable, curable fluid such as a curable adhesive selectedfrom the group consisting of: ultra-violet-curable and otherlight-curable adhesives; heat, two-part, or moisture activatedadhesives; and cyanoacrylate adhesives. In some embodiments, the sealingliquid can be selected from the group consisting of mineral oil,silicone oil, fluorinated oils, and other fluids which are substantiallynon-miscible with water.

In some embodiments, the sealing liquid can be a fluid when it isapplied to the surface of the microplate and in some embodiments, thesealing liquid can remain fluid throughout an analytical or chemicalreaction using the microplate. In some embodiments, the sealing liquidcan become a solid or semi-solid after it is applied to the surface ofthe microplate.

Other Amplification Methods

As should be appreciated from the discussion above, the presentteachings can find utility in a wide variety of amplification methods,such as PCR, Reverse Transcription PCR (RT-PCR), Ligation Chain Reaction(LCR), Nucleic Acid Sequence Based Amplification (NASBA), self-sustainedsequence replication (3SR), strand displacement activation (SDA), Q(3replicase) system, isothermal amplification methods, and other knownamplification method or combinations thereof. Additionally, the presentteachings can find utility for use in a wide variety of analyticaltechniques, such as ELISA; DNA and RNA hybridizations; antibody titerdeterminations; gene expression; recombinant DNA techniques; hormone andreceptor binding analysis; and other known analytical techniques. Stillfurther, the present teachings can be used in connection with suchamplification methods and analytical techniques using not onlyspectrometeric measurements, such as absorption, fluorescence,luminescence, transmission, chemiluminescence, and phosphorescence, butalso colorimetric or scintillation measurements or other known detectionmethods. It should also be appreciated that the present teachings may beused in connection with microcards and other principles, such as setforth in U.S. Pat. Nos. 6,126,899 and 6,124,138.

In some embodiments, the reagents can comprise first and secondoligonucleotides effective to bind selectively to adjacent, contiguousregions of target DNA and that can be ligated covalently by a ligaseenzyme or by chemical means. Such oligonucleotide ligation assays (OLA)are described, for example, in U.S. Pat. No. 4,883,750; and Landegren,U., et al., Science 241:1077 (1988). In this approach, the twooligonucleotides (oligonucleotides) are reacted with the target underconditions effective to ensure specific hybridization of theoligonucleotides to their targets. When the oligonucleotides havebase-paired with their targets, such that confronting end subunits inthe oligonucleotides are base paired with immediately contiguous basesin the target, the two oligonucleotides can be joined by ligation, e.g.,by treatment with ligase. After the ligation step, microplate 20 isheated to dissociate unligated detection probes, and the presence ofligated, target-bound detection probe is detected by reaction with anintercalating dye or by other means. The oligonucleotides for OLA canalso be designed to bring together a fluorescer-quencher pair, asdiscussed above, leading to a decrease in a fluorescence signal when theanalyte sequence is present. In some embodiments of the OLA ligationmethod, the concentration of a target region from an analytepolynucleotide can be increased, if desired, by amplification withrepeated hybridization and ligation steps. Simple additive amplificationcan be achieved using the analyte polynucleotide as a target andrepeating denaturation, annealing, and ligation steps until a desiredconcentration of the ligated product is achieved.

In other embodiments, the ligated product formed by hybridization andligation can be amplified by ligase chain reaction (LCR). In thisapproach, two complementary sets of sequence-specific oligonucleotidedetection probes are employed for each target DNA. One of the two setsof sequence-specific oligonucleotide detection probes comprises firstand second oligonucleotides designed for sequence-specific binding toadjacent, contiguous regions of a first strand of target DNA. The secondof the two sets of sequence-specific oligonucleotide detection probescomprises first and second oligonucleotides designed forsequence-specific binding to adjacent, contiguous regions of a secondstrand of target DNA. With continued cycles of denaturation,reannealing, and ligation in the presence of the two complementaryoligonucleotide sets, the target DNA is amplified exponentially,allowing small amounts of target DNA to be detected and/or amplified. Ina further modification, the oligonucleotides for OLA or LCR assay bindto adjacent regions in a target that are separated by one or moreintervening bases, and ligation is effected by reaction with (i) a DNApolymerase, to fill in the intervening single stranded region withcomplementary nucleotides, and (ii) a ligase enzyme to covalently linkthe resultant bound oligonucleotides.

Detection Probes

In some embodiments, a detection probe comprises a moiety thatfacilitates detection of a nucleic acid sequence, and in someembodiments, quantifiably. In some embodiments, a detection probe cancomprise, for example, a fluorophore such as a fluorescent dye, a haptensuch as a biotin or a digoxygenin, a radioisotope, an enzyme, or anelectrophoretic mobility modifier. In some embodiments, the level ofamplification can be determined using a fluorescently labeledoligonucleotide. In some embodiments, a detection probe can comprise afluorophore further comprising a fluorescence quencher.

In some embodiments, a detection probe can comprise a fluorophore andcan be, for example, a 5′-exonuclease assay probe such as a TaqMan®probe (marketed by Applied Biosystems), a stem-loop Molecular Beacon(see, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517, Nature Biotechnology14:303-308 (1996); Vet et al., Proc Natl Acad Sci USA. 96:6394-6399(1999)), a stemless or linear molecular beacon (see., e.g., PCT PatentPublication No. WO 99/21881), a Peptide Nucleic Acid (PNA) MolecularBeacon™ (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), a linearPNA Molecular Beacon (see, e.g., Kubista et al., SPIE 4264:53-58(2001)), a flap endonuclease probe (see, e.g., U.S. Pat. No. 6,150,097),a Sunrise®/Amplifluor® probe (see, e.g., U.S. Pat. No. 6,548,250), astem-loop and duplex Scorpion™0 probe (see, e.g., Solinas et al.,Nucleic Acids Research 29:E96 (2001), and U.S. Pat. No. 6,589,743), abulge loop probe (see, e.g., U.S. Pat. No. 6,590,091), a pseudo knotprobe (see, e.g., U.S. Pat. No. 6,589,250), a cyclicon (see, e.g., U.S.Pat. No. 6,383,752), an MGB Eclipse™ probe (Marketed by EpochBiosciences), a hairpin probe (see, e.g., U.S. Pat. No. 6,596,490), apeptide nucleic acid (PNA) light-up probe, a self-assembled nanoparticleprobe, or a ferrocene-modified probe described, for example, in U.S.Pat. No. 6,485,901; Mhlanga et al., Methods 25:463-471 (2001); Whitcombeet al., Nature Biotechnology 17:804-807 (1999); Isacsson et al.,Molecular Cell Probes 14:321-328 (2000); Svanvik et al., Anal. Biochem.281:26-35 (2000); Wolffs et al., Biotechniques 766:769-771 (2001),Tsourkas et al., Nucleic Acids Research 30:4208-4215 (2002); Riccelli etal., Nucleic Acids Research 30:4088-4093 (2002); Zhang et al., Sheng WuHua Xue Yu Sheng Wu Li Xue Bao (Shanghai) (Acta Biochimica et BiophysicaSinica) 34:329-332 (2002); Maxwell et al., J. Am. Chem. Soc.124:9606-9612 (2002); Broude et al., Trends Biotechnol. 20:249-56(2002); Huang et al., Chem Res. Toxicol. 15:118-126 (2002); Yu et al.,J. Am. Chem. Soc 14:11155-11161 (2001). In some embodiments, a detectionprobe can comprise a sulfonate derivative of a fluorescent dye, aphosphoramidite form of fluorescein, or a phosphoramidite forms of CY5.Detection probes among those useful herein are also disclosed, forexample, in U.S. Pat. Nos. 5,188,934, 5,750,409, 5,847,162, 5,853,992,5,936,087, 5,986,086, 6,020,481, 6,008,379, 6,130,101, 6,140,500,6,140,494, 6,191,278, and 6,221,604. Energy transfer dyes among thoseuseful herein include those described in U.S. Pat. Nos. 5,728,528,5,800,996, 5,863,727, 5,945,526, 6,335,440, 6,849745, U.S. PatentApplication Publication No. 2004/0126763 A1, PCT Publication No. WO00/13026A1, PCT Publication No. WO 01/19841A1, U.S. Patent ApplicationSer. No. 60/611,119, filed Sep. 16, 2004, and U.S. patent applicationSer. No. 10/788,836, filed Feb. 26, 2004. In some embodiments, adetection probe can comprise a fluorescence quencher such as a blackhole quencher (marketed by Metabion International AG), an Iowa Black™quencher (marketed by Integrated DNA Technologies), a QSY quencher(marketed by Molecular Probes), and Dabsyl and Eclipse™ Dark Quenchers(marketed by Epoch).

In some embodiments, a detection probe can comprise a fluorescent dye.In such embodiments, the fluorescent dye can comprise at least one ofrhodamine green (R110), 5-carboxyrhodamine, 6-carboxyrhodamine,N,N′-diethyl-2′,7′-dimethyl-5-carboxy-rhodamine (5-R6G),N,N′-diethyl-2′,7′-dimethyl-6-carboxyrhodamine (6-R6G),5-carboxy-2′,4′,5′,7′-4,7-hexachlorofluorescein,6-carboxy-2′,4′,5′,7′,4,7-hexachloro-fluorescein,5-carboxy-2′,7′-dicarboxy-4′,5′-dichlorofluorescein,6-carboxy-2′,7′-dicarboxy-4′,5′-dichlorofluorescein,5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,1′,2′-benzo-4′-fluoro-7′,4,7-trichloro-5-carboxyfluorescein,1′,2′-benzo-4′-fluoro-7′,4,7-trichloro-6-carboxy-fluorescein,1′,2′,7′,8′-dibenzo-4,7-dichloro-5-carboxyfluorescein, or those dyes setforth in Table 5.

TABLE 5 Absorbance Emission Extinction Fluorescent Dye (nm) (nm)Coefficient 5-Fluorescein¹ 495 520 73000 5-Carboxyfluorescein (5-FAM ™)¹495 520 83000 6-Carboxyfluorescein (6-FAM ™)¹ 495 520 830006-Carboxyhexachlorofluorescein (6- 535 556 73000 HEX ™)¹6-Carboxytetrachlorofluorescein (6- 521 536 73000 TET ™)¹ JOE ™¹ 520 54873000 LightCycler ® Red 640² 625 640 LightCycler ® Red 705² 685 705Oregon Green ® 488¹ 496 516 76000 Oregon Green ® 500¹ 499 519 84000Oregon Green ® 514¹ 506 526 85000 BODIPY ® FL-X¹ 504 510 70000 BODIPY ®FL¹ 504 510 70000 BODIPY ®-TMR-X¹ 544 570 56000 BODIPY ® R6G¹ 528 54770000 BODIPY ® 650/665¹ 650 665 101000 BODIPY ® 564/570¹ 563 569 142000BODIPY ® 581/591¹ 581 591 136000 BODIPY ® TR-X¹ 588 616 68000 BODIPY ®630/650¹ 625 640 101000 BODIPY ® 493/503¹ 500 509 790005-Carboxyrhodamine 6G¹ 524 557 102000 5(6)-Carboxytetramethylrhodamine546 576 90000 (TAMRA)¹ 6-Carboxytetramethylrhodamine 544 576 90000(TAMRA)¹ 5(6)-Carboxy-X-Rhodamine (ROX)¹ 576 601 820006-Carboxy-X-Rhodamine (ROX)¹ 575 602 82000 AMCA-X (Coumarin)¹ 353 44219000 Texas Red ®-X¹ 583 603 116000 Rhodamine Red ™-X¹ 560 580 129000Marina Blue ®¹ 362 459 19000 Pacific Blue ™¹ 416 451 37000 RhodamineGreen ™-X¹ 503 528 74000 7-diethylaminocoumarin-3- 432 472 56000carboxylic acid¹ 7-methoxycoumarin-3-carboxylic 358 410 26000 acid¹Cy3 ®³ 552 570 150000 Cy3B ®³ 558 573 130000 Cy5 ®³ 643 667 250000Cy5.5 ®³ 675 694 250000 DY-505⁴ 505 530 85000 DY-550⁴ 553 578 122000DY-555⁴ 555 580 100000 DY-610⁴ 606 636 140000 DY-630⁴ 630 655 120000DY-633⁴ 630 659 120000 DY-636⁴ 645 671 120000 DY-650⁴ 653 674 77000DY-675⁴ 674 699 110000 DY-676⁴ 674 699 84000 DY-681⁴ 691 708 125000DY-700⁴ 702 723 96000 DY-701⁴ 706 731 115000 DY-730⁴ 734 750 113000DY-750⁴ 747 776 45700 DY-751⁴ 751 779 220000 DY-782⁴ 782 800 102000Cy3.5 ®³ 581 596 150000 EDANS¹ 336 490 5700 WellRED D2-PA⁵ 750 770170000 WellRED D3-PA⁵ 685 706 224000 WellRED D4-PA⁵ 650 670 203000Pyrene 341 377 43000 Cascade Blue ™¹ 399 423 30000 Cascade Yellow ™¹ 409558 24000 PyMPO¹ 415 570 26000 Lucifer Yellow¹ 428 532 11000 NBD-X¹ 466535 22000 Carboxynapthofluorescein¹ 598 668 42000 Alexa Fluor ® 350¹ 346442 19000 Alexa Fluor ® 405¹ 401 421 35000 Alexa Fluor ® 430¹ 434 54116000 Alexa Fluor ® 488¹ 495 519 71000 Alexa Fluor ® 532¹ 532 554 81000Alexa Fluor ® 546¹ 556 573 104000 Alexa Fluor ® 555¹ 555 565 150000Alexa Fluor ® 568¹ 578 603 91300 Alexa Fluor ® 594¹ 590 617 73000 AlexaFluor ® 633¹ 632 647 100000 Alexa Fluor ® 647¹ 650 665 239000 AlexaFluor ® 660¹ 663 690 132000 Alexa Fluor ® 680¹ 679 702 184000 AlexaFluor ® 700¹ 702 723 192000 Alexa Fluor ® 750¹ 749 775 240000 Oyster556 ®⁶ 556 570 155000 Oyster 645 ®⁶ 645 666 250000 Oyster 656 ®⁶ 656 674220000 5(6)-Carboxyeosin¹ 521 544 95000 Erythrosin¹ 529 544 90000¹Marketed by Molecule Probes; ²Marketed by Roche Applied Science;³Marketed by Amersham Biosciences; ⁴Marketed by Synthegen, LLC;⁵Marketed by Beckman Coulter, Inc.; ⁶Marketed by Denovo Biolabels;

In some embodiments, amplified sequences can be detected indouble-stranded form by a detection probe comprising an intercalating ora crosslinking dye, such as ethidium bromide, acridine orange, or anoxazole derivative, for example, SYBR Green® (marketed by MolecularProbes, Inc.), which exhibits a fluorescence increase or decrease uponbinding to double-stranded nucleic acids. In some embodiments, adetection probe comprises SYBR Green® or Pico Green® (marketed byMolecular Probes, Inc.).

In some embodiments, a detection probe can comprise an enzyme that canbe detected using an enzyme activity assay. An enzyme activity assay canutilize a chromogenic substrate, a fluorogenic substrate, or achemiluminescent substrate. In some embodiments, the enzyme can be analkaline phosphatase, and the chemiluminescent substrate can be(4-methoxyspiro[1,2-dioxetane-3,2′(5′-chloro)-tricyclo[3.3.1.13,7]decan]-4-yl)phenylphosphate. In some embodiments, a chemiluminescentalkaline phosphatase substrate can be CDP-Star® chemiluminescentsubstrate or CSPD® chemiluminescent substrate (marketed by AppliedBiosystems).

In some embodiments, the present teachings can employ any of a varietyof universal detection approaches involving real-time PCR and relatedapproaches. For example, the present teachings contemplate embodimentsin which an encoding ligation reaction is performed in a first reactionvessel (such as for example, an eppendorf tube), and a plurality ofdecoding reactions are then performed in microplate 20 described herein.For example, a multiplexed oligonucleotide ligation reaction (OLA) canbe performed to query a plurality of target DNA, wherein each of theresulting reaction products is encoded with, for example, a primerportion, and/or, a universal detection portion. By including a distinctprimer pair in each of plurality of wells 26 of microplate 20corresponding to the primers sequences encoded in the OLA, a givenencoded target DNA can be amplified by that distinct primer pair in agiven well of plurality of wells 26. Further, a universal detectionprobe (such as, for example, a nuclease cleavable TaqMan® probe) can beincluded in each of plurality of wells 26 of microplate 20 to providefor universal detection of a single universal detection probe. Suchapproaches can result in a universal microplate 20, with its attendantbenefits including, among other things, one or more of economies ofscale, manufacturing, and/or ease-of-use. The nature of the multiplexedencoding reaction can comprise any of a variety of techniques, includinga multiplexed encoding PCR pre-amplification or a multiplexed encodingOLA. Further, various approaches for encoding a first sample with afirst universal detection probe, and a second sample with a seconduniversal detection probe, thereby allowing for two sample comparisonsin a single microplate 20 , can also be performed according to thepresent teachings. Illustrative embodiments of such encoding anddecoding methods can be found for example in PCT Publication No.WO2003US0029693 to Aydin et al., PCT Publication No. WO2003US0029967 toAndersen et al., U.S. Provisional Application Nos. 60/556157 and60/630681 to Chen et al., U.S. Provisional Application No. 60/556224 toAndersen et al., U.S. Provisional Application No. 60/556162 to Livak etal., and U.S. Provisional Application No. 60/556163 to Lao et al.

Single Nucleotide Polymorphism (SNP)

In some embodiments, the detection probes can be suitable for detectingsingle nucleotide polymorphisms (SNPs). A specific example of suchdetection probes comprises a set of four detection probes that areidentical in sequence but for one nucleotide position. Each of the fourdetection probes comprises a different nucleotide (A, G, C, and T/U) atthis position. The detection probes can be labeled with probe labelscapable of producing different detectable signals that aredistinguishable from one another, such as different fluorophores capableof emitting light at different, spectrally resolvable wavelengths (e.g.,4-differently colored fluorophores). In some embodiments, for exampleSNP analysis, two colors can be used for two known variants.

In some embodiments, at least one of the forward primer and the reverseprimer can further comprise a detection probe. A detection probe (or itscomplement) can be situated within the forward primer between the firstprimer sequence and the sequence complementary to the target DNA, orwithin the reverse primer between the second primer sequence and thesequence complementary to the target DNA. A detection probe can compriseat least about 10 nucleotides up to about 70 nucleotides and, moreparticularly, about 15 nucleotides, about 20 nucleotides, about 30nucleotides, about 50 nucleotides, or about 60 nucleotides. In someembodiments, a detection probe (or its complement) can further comprisea Zip-Code™ sequence (marketed by Applied Biosystems). In someembodiments, a detection probe can comprise an electrophoretic mobilitymodifier, such as a nucleobase polymer sequence that can increase thesize of a detection probe, or in some embodiments, a non-nucleobasemoiety that increases the frictional coefficient of the detection probe,such as those mobility modifier described in commonly-owned U.S. Pat.Nos. 5,514,543, 5,580,732, 5,624,800, and 5,470,705 to Grossman. Adetection probe comprising a mobility modifier can exhibit a relativemobility in an electrophoretic or chromatographic separation medium thatallows a user to identify and distinguish the detection probe from othermolecules comprised by the sample. In some embodiments, a detectionprobe comprising a sequence complementary to a detection probe and anelectrophoretic mobility modifier can be, for example, a ZipChute™detection probe (marketed by Applied Biosystems). In these embodiments,hybridization of a detection probe with an amplicon, followed byelectrophoretic analysis, can be used to determine the identity andquantity of the target DNA.

RT-PCR

In some embodiments, the present teaching provide methods and apparatusfor Reverse Transcriptase PCR (RT-PCR), which include the amplificationof a Ribonucleic Acid (RNA) target. In some embodiments, assay 1000 cancomprise a single-stranded RNA target, which comprises the sequence tobe amplified (e.g., an mRNA), and can be incubated in the presence of areverse transcriptase, two primers, a DNA polymerase, and a mixture ofdNTPs suitable for DNA synthesis. During this process, one of theprimers anneals to the RNA target and can be extended by the action ofthe reverse transcriptase, yielding an RNA/cDNA doubled-stranded hybrid.This hybrid can be then denatured and the other primer anneals to thedenatured cDNA strand. Once hybridized, the primer can be extended bythe action of the DNA polymerase, yielding a double-stranded cDNA, whichthen serves as the double-stranded target for amplification through PCR,as described herein. RT-PCR amplification reactions can be carried outwith a variety of different reverse transcriptases, and in someembodiments, a thermostable reverse-transcriptions can be used. Suitablethermostable reverse transcriptases can comprise, but are not limitedto, reverse transcriptases such as AMV reverse transcriptase, Mu LV, andTth reverse transcriptase.

Amplifications for MicroRNA and Small Interfering RNA

In some embodiments, assay 1000 can be an assay for the detection ofRNA, including small RNA. Detection of RNA molecules can be, in variouscircumstances, very important to molecular biology, in research,industrial, agricultural, and clinical settings. Among the types of RNAthat are of interest in some embodiments are, for example, naturallyoccurring and synthetic regulatory RNAs such as small RNA molecules(Lee, et al., Science 294: 862-864, 2001; Ruvkun, Science 294: 797-799;Pfeffer et al., 304: Science 734-736, 2004; Ambros, Cell 107: 823-826,2001; Ambros et al., RNA 9: 277-279, 2003; Carrington and Ambros,Science 301: 336-338, 2003; Reinhart et al., Genes Dev. 16: 1616-1626,2002 Aravin et al., Dev. Cell 5: 337-350, 2003, Tuschel et al., Science294: 853-858, 2001; Susi P. et al., Plant Mol. Biol. 54: 157-174, 2004;Xie et al., PLoS Biol. 2: E104, 2004). Small RNA molecules, such as, forexample, micro RNAs (miRNA), short interfering RNAs (siRNA), smalltemporal RNAs (stRNA) and short nuclear RNAs (snRNA), can be, typically,less than about 40 nucleotides in length and can be of low abundance ina cell. With appropriate detection probes, high-density sequencedetection system 10 can detect miRNA expression found in, for instance,cell samples taken at different stages of development. In someembodiments, coexpression patterns can be analyzed across microplate 20with TaqMan sensitivity, specificity, and dynamic range. In someembodiments, such methods obviate the need for running further assays tovalidate the expression levels. In some embodiments, high-densitysequence detection system 10 can be used to validate that siRNAmolecules have successfully, post-translationally regulated the geneexpression patterns of interest. In some embodiments, such methods maybe useful during the manipulation of gene expression patterns usingsiRNAs in order to elucidate gene function and/or interrelationshipsamongst genes. In some embodiments, gene expression patterns can beintroduced into living cells, cellular assays can be seen onhigh-density sequence detection system 10 and can reveal gene functions.In some embodiments, analysis for small RNA can be run on high-densitysequence detection system 10 allowing for a high number of simultaneousassays 1000 on a single sample with performance that obviates the needfor secondary assays to validate the gene expression results.

In some embodiments, the methods of the present teachings can includeforming a detection mixture comprising a detection probe set ligationsequence, and a primer set. In such embodiments, any detection probe setligation sequence comprised by the detection mixture can be amplifiedusing PCR on high-density sequence detection system 10 and thereby forman amplification product. In such embodiments, detection ofamplification of any detection probe ligation sequence of an analyte. Insome embodiments, detection of amplification by high-density sequencedetection system 10 can comprise detection of binding of a detectionprobe to a detection probe hybridization sequence comprised by a probeset ligation sequence or an amplification product thereof. In someconfigurations, detecting can comprise contacting a PCR amplificationproduct such as an amplified probe set ligation sequence with adetection probe comprising a label under hybridizing conditions.

Pre-Amplification and Multiplex Methods

In some embodiments for amplification of a polynucleotide, assay 1000can comprise a preamplification product, wherein one or morepolynucleotides in an analyte has been amplified prior to beingdeposited in at least one of the plurality of wells 26. In someembodiments, these methods can further comprise forming a plurality ofpreamplification products by subjecting an initial analyte comprising aplurality of polynucleotides to at least one cycle of PCR to form adetection mixture comprising a plurality of preamplification products.The detection mixture of preamplification products can be then used forfurther amplification using microplate 20 and high-density sequencedetection system 10. In some embodiments, preamplification comprises theuse of isothermal methods.

In some embodiments, a two-step multiplex amplification reaction can beperformed wherein the first step truncates a standard multiplexamplification round to boost a copy number of the DNA target by about100-1000 or more fold. Following the first step, the resulting productcan be divided into optimized secondary single amplification reactions,each containing one or more of the primer sets that were used previouslyin the first or multiplexed booster step. The booster step can occur,for example, using an aqueous target or using a solid phase archivednucleic acid. See, for example, U.S. Pat. No. 6,605,452, Marmaro.

In some embodiments, preamplification methods can employ in vitrotranscription (IVT) comprising amplifying at least one sequence in acollection of nucleic acids sequences. The processes can comprisesynthesizing a nucleic acid by hybridizing a primer complex to thesequence and extending the primer to form a first strand complementaryto the sequence and a second strand complementary to the first strand.The primer complex can comprise a primer complementary to the sequenceand a promoter region in anti-sense orientation with respect to thesequence. Copies of anti-sense RNA can be transcribed off the secondstrand. The promoter region, which can be single or double stranded, canbe capable of inducing transcription from an operably linked DNAsequence in the presence of ribonucleotides and a RNA polymerase undersuitable conditions. Suitable promoter regions may be prokaryoteviruses, such as from T3 or T7 bacteriophage. In some embodiments, theprimer can be a single stranded nucleotide of sufficient length to actas a template for synthesis of extension products under suitableconditions and can be poly (T) or a collection of degenerate sequences.In some embodiments, the methods involve the incorporation of an RNApolymerase promoter into selected cDNA molecule by priming cDNAsynthesis with a primer complex comprising a synthetic oligonucleotidecontaining the promoter. Following synthesis of double-stranded cDNA, apolymerase generally specific for the promoter can be added, andanti-sense RNA can be transcribed from the cDNA template. Theprogressive synthesis of multiple RNA molecules from a single cDNAtemplate results in amplified, anti-sense RNA (aRNA) that serves asstarting material for cloning procedures by using random primers. Theamplification, which will typically be at least about 20-40, typicallyto 50 to 100 or 250-fold, but can be 500 to 1000-fold or more, can beachieved from nanogram quantities or less of cDNA.

In some embodiments, a two stage preamplification method can be used topreamplify assay 1000 in one vessel by IVT and, for example, thispreamplification stage can be 100×sample. In the second stage, thepreamplified product can be divided into aliquots and preamplified byPCR and, for example, this preamplification stage can be 16,000×sampleor more. Although the above preamplification methods can be used inmicroplate 20, these are only examples and are non-limiting.

In some embodiments, the preamplification can be a multiplexpreamplification, wherein the analyte sample can be divided into aplurality of aliquots. Each aliquot can then be subjected topreamplification using a plurality of primer sets for DNA targets. Insome embodiments, the primer sets in at least some of the plurality ofaliquots differ from the primer sets in the remaining aliquots. Eachresulting preamplification product detection mixture can then bedispersed into at least some of the plurality of wells 26 of microplate20 comprising an assay 1000 having corresponding primer sets anddetection probes for further amplification and detection according tothe methods described herein. In some embodiments, the primer sets ofassay 1000 in each of the plurality of wells 26 can correspond to theprimer sets used in making the preamplification product detectionmixture. The resulting assay 1000 in each of the plurality of wells 26thus can comprise a preamplification product and primer sets anddetection probes for amplification for DNA targets, which, if present inthe analyte sample, have been preamplified.

Since a plurality of different sequences can be amplified simultaneouslyin a single reaction, the multiplex preamplification can be used in avariety of contexts to effectively increase the concentration orquantity of a sample available for downstream analysis and/or assays. Insome embodiments, because of the increased concentration or quantity oftarget DNA, significantly more analyses can be performed with multiplexamplified samples than can be performed with the original sample. Inmany embodiments, multiplex amplification further permits the ability toperform analyses that require more sample or a higher concentration ofsample than was originally available. In such embodiments, multiplexamplification enables downstream analysis for assays that could not havebeen possible with the original sample due to its limited quantity. Insome embodiments, the plurality of aliquots can comprise 16 aliquotswith each of the 16 aliquots comprising about 1536 primer sets. In suchembodiments, a sample comprising a whole genome for a species, forexample a human genome, can be preamplified. In some embodiments, theplurality of aliquots can be greater than 16 aliquots. In someembodiments, the number of primer sets can be greater than 1536 primersets. In some embodiments, the plurality of aliquots can be less than 16aliquots and the number of primer sets can be greater than 1536 primersets. For examples of such embodiments, see PCT Publication No. WO2004/051218 to Andersen and Ruff.

Multiplex Methods

In some embodiments, multiplex methods are provided wherein assay 1000comprises a first universal primer that binds to a complement of a firsttarget, a second universal primer that binds to a complement of a secondtarget, a first detection probe comprising a sequence that binds to thesequence comprised by the first target, and a second detection probecomprising a sequence that binds to a sequence comprised by the secondtarget. In some embodiments, at least some of the plurality of wells 26of microplate 20 comprise a solution operable to perform multiplex PCR.The first and second detection probes can comprise different labels, forexample, different fluorophores such as, in non-limiting example, VICand FAM. Sequences of the first and second detection probes can differby as little as one nucleotide, two nucleotides, three nucleotides, fournucleotides, or greater, provided that hybridization occurs underconditions that allow each detection probe to hybridize specifically toits corresponding detection probe.

In some embodiments, multiplex PCR can be used for relativequantification, where one primer set and detection probe amplifies thetarget DNA and another primer set and detection probe amplifies anendogenous reference. In some embodiments, the present teaching providefor analysis of at least four DNA targets in each of the plurality ofwells 26 and/or analysis of a plurality of DNA targets and a referencein each of the plurality of wells 26.

Kits

In some embodiments, kits can be provided comprising materials suitablefor carrying out polynucleotide amplification. In some embodiments, suchkits can comprise microplate 20 and at least a master mix, such asdescribed above herein.

In some embodiments, such kits can comprise solutions packaged forpreamplification of targets for downstream or subsequent analysisincluding by multiplex PCR. In some embodiments, the kits can comprise aplurality of primer sets. In some embodiments, the kits can furthercomprise a set of amplification primers suitable for pre-amplifying asample of target DNA disposed in at least some of the plurality of wells26. In some embodiments, the primers comprised in each of the pluralityof wells 26 can, independently of one another, be the same or adifferent set of primers.

In some embodiments, the kit can comprise at least one primer and atleast one detection probe disposed in at least some of the plurality ofwells 26. In some embodiments, the kit can comprise a forward primer, areverse primer, and at least one FAM labeled MGB quenched PCR detectionprobe disposed in at least some of the plurality of wells 26. In someembodiments, the kit can comprise at least one detection probe, at leastone primer, and a polymerase. In some embodiments, the kit can compriseat least one forward primer, at least one reverse primer, at least onelabeled MGB quenched detection probe, at least one labeled MGB quencheddetection probe used as a endogenous control, and a polymerase disposedin at least some of the plurality of wells 26. In some embodiments, aROX labeled detection probe can be used as a passive internal reference.Some embodiments comprise other detection probes to be used as a passiveinternal reference. In some embodiments, the kit can comprise reagentsfor preamplification. In some embodiments, reaction vessels, separatefrom microplate 20, can contain any of the above reagents in a driedform, which can be coated to or directed to the bottom of at least someof the plurality of wells 26. In some embodiments, the user can add auniversal master mix, water, and a sample of target DNA to each of theplurality of wells 26 before analysis.

In some embodiments, a kit comprises a container containing assayreagents and a separate data storage medium that contains data about theassay reagents. The assay reagents can be adapted to perform an allelicdiscrimination or expression analysis reaction when mixed with at leastone target polynucleotide. The other reagents can be, for example,components conventionally used for PCR and can comprise non-reactivecomponents. In some embodiments, the assay reagents container can have amachine-readable label that provides information about the contents ofthe container.

In some embodiments, the data stored on the data storage medium cancomprise computer-readable code that can be used to adjust, calibrate,direct, set, run, or otherwise control an apparatus, for example,high-density sequence detection system 10. In some embodiments, the datastored on the date storage medium can be used to control high-densitysequence detection system 10 to automatically perform PCR or RT-PCR ofassay 1000. See, for example, U.S. Patent Application Publication No.2004/0072195.

Data Analysis

In some embodiments, as seen in FIG. 58, a plurality of microplates 20having assay 1000 filled thereon can be analyzed as described hereinwith high-density sequence detection system 10 to generate data. In someembodiments, this data can be stored in a gene expression analysissystem database 736. Software can then be used to generate geneexpression analysis information 738.

In some embodiments, a gene expression analysis system can utilizecomputer software that organizes analysis sessions into studies andstores them in database 738. An analysis session can comprise theresults of running microplate 20 in high-density sequence detectionsystem 10. To analyze session data, one can load an existing study thatcontains analysis session data or create a new study and attach analysissession data to it. Studies can be opened and reexamined an unlimitednumber of times to reanalyze the analysis session data or to add otheranalysis sessions to the analysis.

In some embodiments, gene expression analysis system database 736 storesthe analyzed data for each microplate 20 run on high-density sequencedetection system 10 as an analysis session in database 736. The softwarecan identify each analysis session by marking indicia 64 of theassociated microplate 20 and the date on which it was created. Onceanalysis sessions have been assigned to a study, various functions canbe performed. These functions comprise, but are not limited to,designating replicates, removing outliers, filtering data out of aparticular view or report, correction of preamplification values viastored values, and computation of gene expression values.

In some embodiments, real time PCR is adapted to perform quantitativereal time PCR (qRT-PCR). In some embodiments, two different methods ofanalyzing data from qRT-PCR experiments can be used: absolutequantification and relative quantification. In some embodiments,absolute quantification can determine an input copy number of the targetDNA of interest This can be accomplished, for example, by relating asignal from a detection probe to a standard curve. In some embodiments,relative quantification can describe the change in expression of thetarget DNA relative to a reference or a group of references such as, foran example, an untreated control, an endogenous control, a passiveinternal reference, an universal reference RNA, or a sample at time zeroin a time course study. When determining absolute quantification, theexpression of the target DNA can be compared across many samples, forexample, from different individuals, from different tissues, frommultiple replicates, and/or serial dilution of standards in one or morematrices. In some embodiments of the present teachings, qRT-PCR can beperformed using relative quantification and the use of standard curve isnot required. Relative quantification can compare the changes in steadystate target DNA levels of two or more genes to each other with one ofthe genes acting as an endogenous reference, which may be used tonormalize a signal from a sample gene. In some embodiments, in order tocompare between experiments, resulting fold differences from thenormalization of sample to the reference can be expressed relative to acalibrator sample. In some embodiments, the calibrator sample isincluded in each assay 1000. The gene expression analysis system candetermine the amount of target DNA, normalized to a reference, bydeterminingΔC _(T) =C _(Tq) −C _(Tendo)where C_(T) is the threshold cycle for detection of a fluorophore inreal time PCR; C_(Tq) is the threshold cycle for detection of afluorophore for a target DNA in assay 1000; and C_(Tendo) is thethreshold cycle for detection of a fluorophore for an endogenousreference or a passive internal reference in assay 1000.

In some embodiments, a gene expression analysis system can determine theamount of target DNA, normalized to a reference and relative to acalibrator, by determining:ΔΔC _(T) =ΔC _(T,q) −ΔC _(T,cb)where C_(T,q) is the threshold cycle for detection of a fluorophore forthe target DNA in assay 1000; C_(T,cb) is the threshold cycle fordetection of a fluorophore for a calibrator sample; ΔC_(T,q) is adifference in threshold cycles for the target DNA and an endogenousreference; and ΔC_(T,cb) is a difference in threshold cycles for thecalibrator sample and the endogenous reference If ΔΔC_(T) is determined,the relative quantity of the target DNA can be determined using arelationship of relative quantity of the target DNA can be equal to2^(−ΔΔCT). In some embodiments, ΔΔC_(T) can be about zero. In someembodiments, ΔΔC_(T) can be less than ±1. In some embodiments, the abovecalculations can be adapted for use in multiplex PCR (See, for example,Livak et al. Applied Biosystems User Bulletin #2, updated October 2001and Livak and Schmittgen, Methods (25) 402-408 (2001).Triple Delta Analysis

In some embodiments, assay 1000 can be preamplified, as discussedherein, in order to increase the amount of target DNA prior todistribution into the plurality of wells 26 of microplate 20. In someembodiments, assay 1000 can be collected, for example, via a needlebiopsy that typically yields a small amount of sample. Distributing thissample across a large number of wells can result in variances in sampledistribution that can affect the veracity of subsequent gene expressioncomputations. In such situations, assay 1000 can be preamplified using,for example, a pooled primer set to increase the number of copies of alltarget DNA simultaneously.

In some embodiments, preamplification processes can be non-biased, suchthat all target DNA are amplified similarly and to about the same power.In such embodiments, each target DNA can be amplified reproducibly fromone input sample to the next input sample. For example, if target DNA Xis initially present in sample A at 100 target molecules, then after 10cycles of PCR amplification (1000-fold), 100,000 target molecules shouldbe present. Continuing with the example, if target DNA X is initiallypresent in sample B at 500 target molecules, then after 10 cycles of PCRamplification (1000-fold), 500,000 target molecules should be present.In this example, the ratio of target DNA X in samples A/B remainsconstant before and after the amplification procedure.

In some embodiments, a minor proportion of all target DNA can have anobserved preamplification efficiency of less than 100%. In suchembodiments, if the amplification bias is reproducible and consistentfrom one input sample to another, then the ability to accurately computecomparative relative quantitation between any two samples containingdifferent relative amounts of target can be maintained. Continuing theexample from above and assuming 50% reproducible amplificationefficiency, if target DNA X is initially present in sample A at 100target molecules, then after 10 cycles of PCR amplification (50% of1000-fold), 50,000 target molecules should be present. Furthercontinuing the example, if target X is initially present in sample B at500 target molecules, then after 10 cycles of PCR amplification (50% of1000-fold), 250,000 target molecules should be present. In this example,the ratio of template X in samples A/B remains constant before and afterthe amplification procedure and is the same ratio as the 100% efficiencyscenario.

In some embodiments, an unbiased amplification of each target DNA (x, y,z, etc.) can be determined by calculating the difference in C_(T) valueof the target DNA (x,y,z, etc.) from the C_(T) value of a selectedendogenous reference, and such calculation is referred to as the ΔC_(T)value for each given target DNA, as described above. In someembodiments, a reference for a bias calculation can be non-preamplified,amplified target DNA and an experimental sample can be a preamplifiedamplified target DNA. In some embodiments, the standard sample andexperimental sample can originate from the same sample, for example,same tissue, same individual, and/or same species. In some embodiments,comparison of ΔC_(T) values between the non-preamplified amplifiedtarget DNA and preamplified amplified target DNA can provide a measurefor the bias of the preamplification process between the endogenousreference and the target DNA (x, y, z, etc.).

In some embodiments, the difference between the two ΔC_(T) values(ΔΔC_(T)) can be zero and as such there is no bias frompreamplification. This is illustrated below with reference to FIG. 213.In some embodiments, the gene expression analysis system can becalibrated for potential differences in preamplification efficiency thatcan arise from a variety of sources, such as the effects of multipleprimer sets in the same reaction. In some embodiments, calibration canbe performed by computing a reference number that reflectspreamplification bias. Reference number similarity for a given targetDNA across different samples is indicative that the preamplificationreaction ΔC_(T)s can be used to achieve reliable gene expressioncomputations.

In some embodiments of the present teaching, a gene expression analysissystem can compute these reference numbers by collecting a sample(designated as Sample A and S_(A)) and processing it with one or moreprotocols. A first protocol comprises running individual PCR geneexpression reactions for each target DNA (T_(x)) relative to anendogenous reference (endo), such as, for example, 18 s or GAPDH. Thesereactions can yield cycle threshold values for each target DNA relativeto the endogenous control; as computed by:ΔC _(T not preamplified) T _(x)S_(A) =C _(T not preamplified) T_(x)S_(A)−C _(T notpreamplified) endo

A second protocol can comprise running a single PCR preamplificationstep on assay 1000 with, for example, a pooled primer set. In someembodiments, the pooled primer set can contain primers for each targetDNA. Subsequently, the preamplified product can be distributed amongplurality of wells 26 of microplate 20. PCR gene-expression reactionscan be run for each preamplified target DNA (Tx) relative to anendogenous reference (endo). These reactions can yield cycle thresholdvalues for each preamplified target DNA relative to the endogenouscontrol, as computed by:ΔC _(T preamplified) T_(x)S_(A) =C _(T preamplified) T _(x)S_(A) −C_(T preamplified) endoA difference between these ΔC_(T not preamplified) T_(x)S_(A) andΔC_(T preamplified) T_(x)S_(A) can be computed by:ΔΔC _(T) T _(x) S _(A) =ΔC _(T not preamplified) T _(x) S _(A) −ΔC_(T preamplified) T _(x) S _(A)

In some embodiments, a value for ΔΔC_(T)T_(x)S_(A) can be zero or closeto zero, which can indicate that there is no bias in thepreamplification of target DNA T_(x). In some embodiments, a negativeΔΔC_(T) T_(x)S_(A) value can indicate the preamplification process wasless than 100% efficient for a given target DNA (T_(x)). For example,when using an IVT process, a percentage of target DNA with a ΔΔC_(T) of+/−1 C_(T) of zero can be ˜50%. In another example, when using amultiplex preamplification process, a percentage of target DNA with aΔΔCT of +/−1 C_(T) of zero can be ˜90%.

In some embodiments, an amplification efficiency can be less than 100%for a particular target DNA, therefore ΔΔC_(T) is less than zero for theparticular target DNA. An example can be an evaluation of ΔΔC_(T) valuesfor a group of target DNA from a 1536-plex for the multiplexpreamplification process including four different human sample inputsources: liver, lung, brain and an universal reference tissue composite.In this example, most ΔΔC_(T) values are near zero, however, some of thetarget DNA have a negative ΔΔC_(T) value but these negative values arereproducible from one sample input source to another. In someembodiments, a gene expression analysis system can determine if a biasexists for target DNA analyzed for different sample inputs.

In some embodiments of the present teachings, a gene expression analysissystem can use ΔΔC_(T) values computed for the same target DNA but indifferent samples (Sample A (S_(A)) and Sample B (S_(B))) in order todetermine the accuracy of subsequent relative expression computations.This results in the equation,ΔΔΔC _(T) T _(x) =ΔΔC _(T) T _(x) S _(A) −ΔΔC _(T) _(x) S _(B)In some embodiments a value for ΔΔΔC_(T)T_(x) can be zero or reasonablyclose to zero which can indicate that the preamplified ΔC_(T) values forT_(x) (ΔC_(T preamplified) T_(x)S_(A) and ΔC_(T preamplified)T_(x)S_(B)) can be used for relative gene expression computation betweendifferent samples via a standard relative gene expression calculation.

In some embodiments, a standard relative gene expression calculation candetermine the amount of the target DNA. In some embodiments, a standardrelative gene expression calculation employs a comparative C_(T). Insome embodiments, the above methods can be practiced during experimentaldesign and once the conditions have been optimized so that theΔΔΔC_(T)T_(x) is reasonably close to zero, subsequent experiments onlyrequire the computation of the ΔC_(T) value for the preamplifiedreactions. In some embodiments, ΔΔC_(T)T_(x)S_(A) values can be storedin a database or other storage medium. In such embodiments, these valuescan then be used to convert ΔΔC_(Tpreamplified)T_(x)S_(A) values toΔΔC_(T not preamplified)T_(x)S_(A) values. In such embodiments, theΔΔC_(T preamplified)T_(x)S_(y) values can be mapped back to a commondomain. In some embodiments, a not preamplified domain can be calculatedusing other gene expression instrument platforms such as, for example, amicroarray. In some embodiments, the ΔΔC_(T)T_(x)S_(A) values need notbe stored for all different sample source inputs (S_(A)) if it can beillustrated that the ΔΔC_(T preamplified)T_(x) is reasonably consistentover different sample source inputs.

In some embodiments, after microarray sample-to-sample differences arein a ΔΔC_(T) format, then real-time PCR data can be directly compared todata from other platforms. In some embodiments, a ΔΔΔC_(T) calculationcan be a validation tool to confirm that relative quantitation data canbe compared from one amplification/detection process to another. In someembodiments, ΔΔΔC_(T) calculation can be a validation tool to confirmthat relative quantitation data can be compared from one sample inputsource to another sample input source, for example, comparing a samplefrom liver to a sample from brain in the same individual. In someembodiments, ΔΔΔC_(T) calculation can be a validation tool to confirmthat relative quantitation data can be compared from one high-densitysequence detector system 10 to another high-density sequence detectionsystem 10. In some embodiments, ΔΔΔC_(T) calculation can be a validationtool to confirm that relative quantitation data can be compared from oneplatform to another, for example, data from real time PCR to data from ahybridization array is especially valuable for cross-platformvalidation. In some embodiments, real time PCR and hybridization arraydata can be directly compared. In some embodiments, the TaqMan ΔΔC_(T)can be compared to a microarray output converted to the ΔΔC_(T) format.In such embodiments, the resultant ΔΔΔC_(T), if within +/−1 C_(T) ofzero, can determine a high-degree of confidence that the actual folddifference observed within each of the two platforms is correlative.

Assay Controls

In some embodiments, high-density sequence detection system 10 measuresthe relative quantities of target DNA using the C_(T) value from a PCRgrowth curve, as described herein. The measured C_(T) value for targetDNA for a given assay may vary depending on the system and/or microplate20 in which the assay 1000 is measured. That is, such variation mayarise from manufacturing differences in high-density sequence detectionsystem 10 and/or thermal non-uniformity from variances in production ofmicroplate 20.

In some embodiments, normalization may be the adjusting of a set of rawmeasurements. For example, a variable storing target DNA levels,quantities may be represented in copy numbers, according to sometransformation function in order to make such data compatible betweendifferent samples. For example, adjusting copy numbers for a target DNAquantity will produce measurements normalized against a quantity oftotal RNA and therefore such data can be expressed in specificmeaningful and/or compatible units. Without relevant normalization, rawmeasurements may not carry information that is easily interpretable.

In some embodiments, several of the plurality of wells 26 of microplate20 can be allocated for controls. In some embodiments, the controlcomprises a template. The template can be, for example, a syntheticoligonucleotide or plasmid, genomic DNA, or other natural DNA or RNA. Insome embodiments, the template can contain analogs of naturallyoccurring nucleotides with modifications to the base, sugar, orphosphate backbone, such as PNAs.

In some embodiments, exogenous templates can be used as controls andsuch templates can be introduced into assay 1000 in one of the followingways:

-   -   (i) the template at a known concentration can be introduced into        a reverse transcription reaction along with the sample;    -   (ii) the template at a known concentration can be introduced        into a preamplification reaction along with the sample;    -   (iii) the template at a known concentration can be introduced        into assay 1000 along with the sample; or    -   (iv) the template at a known concentration can be spotted onto        at least one of a plurality of wells 26.

In some embodiments, the exogenous template can be spotted and driedinto at least some of the plurality of wells 26 at a known and definedconcentration and the C_(T) value measured from those of the pluralityof wells 26 comprising the control. This C_(T) value can be used tocorrect for high-density sequence detection system 10, microplate 20,and sample filling/pipetting variations. In these embodiments, assay1000 can be used to fill at least some of the plurality of wells 26, butassay 1000 would not contain any exogenous template that would beamplified. In some embodiments, the template can be filled into at leastsome of the plurality of wells 26 at a known and defined concentrationand the C_(T) value can be measured from the plurality of wells 26comprising the control to correct for variations from sample filling andpipetting. Templates can also be detected in some of the plurality ofwells 26 as an internal control. In such embodiments, the detectionprobe for the template would produce a different signal than thedetection probe for the target DNA. In some embodiments that include apreamplification method to amplify targets prior to PCR, the templatecan also be designed such that it can be preamplified. Thus, if thetemplate is introduced to assay 1000 prior to preamplification andsubsequently measured on microplate 20, its C_(T) value could be used tocorrect for variations in the efficiency of sample preamplification aswell as filling/pipetting errors.

In some embodiments, the plurality of wells 26 used for controls onmicroplate 20 can be allocated to contain at least one fluorescent dyethat can be spotted and dried down into microplate 20 and hydrolyzed atthe time of sample filling. Such plurality of wells 26 can be used toimprove calibration of detection system 300 for optical aberrations. Insome embodiments, a dye can be used at known concentration and thesignals therefrom can be used to optimize the detection sensitivity ofhigh-density sequence detection system 10 (such as the exposure time ofthe CCD in a detection system 300). In some embodiments, the pluralityof wells 26 comprising a series dilution of control wells can be usedfor such calibrations and optimizations. In some embodiments, some ofthe plurality of wells 26 can be used as controls for identification ofthe position of the plurality of wells 26. In some embodiments, at leastsome of the plurality of wells 26 on microplate 20 can comprise apassive internal reference dye (PIR), such as for example, ROX. Thesignal from the PIR can be used to locate the plurality of wells 26 bydetection system 300. In some embodiments, prior to beginning PCR,background signals from quenching dyes can be used to determine thelocations of the plurality of wells 26 by detection system 300. In someembodiments, controls can be used to determine filling errors. That is,signals from the PIR can be used to determine if sample filling errorshave occurred by looking for an absent or an abnormally high or lowsignal in the PIR detection image or channel. These signals can indicatean empty well, or an overfilled or under filled well, respectively. Insome embodiments, controls can be used to determine spotting errors. Thebackground signals from quenching dyes can be used to determine ifspotting errors occurred by looking for an absent or an abnormally highor low signal in the quenching detection image or channel.

In some embodiments, controls can be used as quality control forspotting reagents onto microplate 20. Controls can be measured (byimaging or scanning) for the weak background fluorescence of the drieddown reagents to determine if the plurality of wells 26 were spottedcorrectly and/or in the correct orientation. In some embodiments, one ormore fluorescent, infrared, ultraviolet, or visible dyes are introducedinto the reagents prior to spotting. When dried down, the fluorescentdyes can be measured to determine if spotting was performed correctly.In some embodiments, the addition of extra dyes to the spotting reagentscan be useful for spotting reagents that do not have an inherentfluorescent signal, such as for example the use of reagents comprisingSYBR® detection probes. In such embodiments, these additional dyes couldalso be used as internal controls for identifying filling and pipettingerrors.

In some embodiments, the plurality of wells 26 without detection probesor primers and/or the plurality of wells 26 that are completely empty orfilled with buffer or other solution not containing dye can be used forbackground correction. The plurality of wells 26 comprising controlswithout templates (no template controls (NTC)) can also be used forbackground correction and/or for confirming lack of contamination of theplurality of wells 26 by other samples. In some embodiments, theplurality of wells 26 comprising controls without assay 1000 can be usedto confirm lack of contamination during spotting. In some embodiments,the plurality of wells 26 containing varying amounts of a single ormultiple dyes can be used to determine if high-density sequencedetection system 10 is capable of detecting signals within the expecteddynamic range independent of assay performance. In some embodiments, theplurality of wells 26 containing varying amounts of a single or multipledyes can be used to correct for optical crosstalk or other means ofsignal correction or normalization. Examples include serial dilutions,multiple titration points, dye ladders, as well as replicates andcombinations thereof. In some embodiments, pin hole arrays are used foroptical calibration. The controls described above, individually or incombinations thereof, can be incorporated into a single microplate 20 tobe used to verify high-density sequence detection system 10 performancein the field at the time of installation or during manufacture.

In some embodiments, a procedure for calibration of spectral sensitivitycan employ a reference standard to apply a correction to a spectrum suchthat each of the plurality of wells 26 signal for each filter isnormalized to a specific value. In some embodiments, the referencestandard can comprise serial dilutions, multiple titration points or dyeladders, as well as replicates and combinations thereof. In someembodiments, the reference comprises multiple dyes (e.g., two, three,four, five, or more) in some of the plurality of wells 26 of microplate20. In some embodiments, the value should be identical across allinstruments and time periods in order to preserve the calibration. Insome embodiments, a reference can be fluorescent reference standard. Insome embodiments, the reference can be used in normalizing a singlehigh-density sequence system 10. In some embodiments, the reference canbe used to normalize a group of high-density sequence systems 10. Insome embodiments, the procedure normalizes thresholds and baselines overa group of high-density sequence detector systems 10 so that C_(T)values are similar across the group for the same assay 1000. In someembodiments, the controls are templates.

In some embodiments, the templates are introduced into a mixturecomprising a sample prior to reverse transcription and the resultingC_(T) values generated from the templates are used to correct forvariations in the efficiency of the reverse transcription reactionrelative to the expected C_(T) value. In some embodiments, templates areintroduced into a mixture comprising a sample prior to preamplificationand the resulting C_(T) values generated from the templates are used tocorrect for variations in efficiency of the preamplification reaction.In some embodiments, the templates are introduced into a mixturecomprising the sample prior to amplification and the resulting C_(T)values generated from the templates are used to correct for variationsin efficiency of amplification. In some embodiments, different templatesare introduced into the mixture comprising a sample at the threedifferent steps (i) reverse transcription, (ii) preamplification and(iii) amplification and the resulting C_(T) values generated from thetemplates are calculated for each of the three steps. In suchembodiments, the resulting C_(T) value generated from the templates canbe used to determine which of the three steps can be responsible forlarge deviations of C_(T) measurements from the expected values.Multiple exogenous templates with varying relative concentrations can beadded to a sample mixture in any of the three steps or all of the steps.In some embodiments, a standard plot for absolute quantitation of asample run on microplate 20 can be calculated. The standard plot can beused to normalize data attained from different microplates 20 or fromdifferent samples on the same microplate 20.

In some embodiments, a control can comprise an endogenous template or aset of endogenous templates within a sample that can be used in a widerange of tissues. In some embodiments, the endogenous template can beselected so that the average signal produced during amplification isconsistent from sample to sample. In some embodiments, the appropriatelyselected endogenous template can be used to normalize for variations insample quantity in the plurality of wells 26. In some embodiments,results from endogenous controls can be compared from results fromexogenous control to distinguish variations in sample quantity andvariations in assay performance. A dataset can be normalized by using afunction of multiple endogenous templates as controls. For example, aregression of the mean expression values from multiple endogenouscontrols and can be chosen to be expressed across the entire expressionrange. Other examples of normalization using a function includefunctions of the mean signal across microplate 20, median normalization,quantile normalization, and lowness normalization. In some embodiments,the endogenous controls are relatively invariantly expressed acrossstandard experimental conditions or biological conditions, for example,a tumor, or non-tumor tissue. In some embodiments, the endogenouscontrols are relatively, invariantly expressed across different tissuetypes, for example, brain and lung. In some embodiments, a singleendogenous control can be used for normalization. In some embodiments,multiple endogenous controls are used for normalization.

In some embodiments, microplate 20 comprising a calibrated dilutionseries of DNA targets and single exon assays can be run on high-densitysequence detection system 10 and the data collected can be used tocalibrate for absolute quantity or copy number estimations or as incomparison to other array platforms. In such embodiments, microplate 20can comprise a combination of replicated bacterial DNA and human DNA.For example, microplate 20 can be spotted with 96 different primer setsand 64 replications of the ten-fold primer sets. The human sample can besplit and then spiked with bacterial targets to make a set of fourten-fold dilutions. Microplate 20 comprising 96 primer sets with 64replications can be filled with the set of four ten-fold dilutions andrun in high-density sequence detection system 10 producing data for 16replications of each dilution of the set. The data collected can be usedfor calculation of high-level performance parameters such as tabulatingbad data, calibrating random error model, estimating systematic errors,and estimating starting copy number.

In some embodiments, controls can be used for spatial normalization thatcompensates between at least two channels of signal that is beingcollected by detections system 300. The channels for which a signal canbe being collected and imaged can be different band passes and theoptical performance can change with wavelength and detection probe. Insome embodiments, spatial normalization can be accomplished bycalibration images of each of the at least two channels collected from amixture of a pure detection probe spotting to the channel. In someembodiments, a control comprising a mixture of dyes can be spotted ontomicroplate. In such embodiments, the control comprising a mixture ofdyes produces a high signal to noise ratio when detected in detectionsystem 300 of high-density sequence detection system 10. In suchembodiments, spatial normalization correction can be calculated by theuse of spatial trends of the measurements of the controls. The controlscomprising a mixed dye can be placed in the grid throughout microplate20. In some embodiments, to correct all extracted normalizationintensities for the spatial trends, a coarse image can be collected andnormalized to a 1, 2D median smoothed inner plated under every featurecollected is then divided into the image of the extracted normalizedintensities. In some embodiments, spatial normalization allows forplatform comparisons of data, removes specific instrument effects, orimproves cross instrument and cross platform comparisons. In someembodiments, any of the controls discussed above can be adapted forgenotyping applications.

Assay Selection and Polynucleotide Library

In some embodiments, a method is provided for supplying a user withassays useful in obtaining structural genomic information, such as thepresence or absence of one or more SNPs, and functional genomicinformation, such as the expression or amount of expression of one ormore genes. As such, in some embodiments, the assays can be configuredto detect the presence or expression of genetic material in the sample.

In some embodiments, a method of compiling a library of polynucleotidedata sets can be provided. In such embodiments, the data sets cancorrespond to polynucleotides that each function as a primer forproducing a nucleic acid sequence that can be complementary to at leastone target SNP, as a detection probe for rendering detectable the atleast one target SNP, or as both. According to some embodiments, themethod can comprise selecting for the library polynucleotide data setsthat each correspond to a respective polynucleotide that contains asequence that is complementary to a respective first allele in each ofthe at least one target, if, under a set of reaction conditions a numberof parameters are met by each polynucleotide corresponding to the datasets in the library.

In some embodiments, the method can comprise determining a backgroundsignal value by calculating a first normalized ratio of a fluorescenceintensity of a respective polynucleotide that contains a sequence thatis complementary to a first allele comprised in the at least one targetnucleic acid sequence, reacted with first assay reactants in the absenceof the target nucleic acid sequence, and under first conditions offluorescence excitation, to a dye fluorescence intensity of apassive-reference dye under the first conditions. The method cancomprise comparing a difference between a second normalized ratio of thefluorescence intensity of the respective polynucleotide reacted with thefirst assay reactants in the presence of the target nucleic acidsequence, to the dye fluorescence intensity, and the background signalvalue. The method can comprise comparing a difference between a thirdnormalized ratio of the fluorescence intensity of the respectivepolynucleotide reacted with second assay reactants that contain a secondallele comprised in the at least one target nucleic acid sequence to thedye fluorescence intensity, wherein the second allele differs from thefirst allele, and the background signal value.

In some embodiments, the method can comprise determining whether atleast one individual from a population of individuals has a genotypeidentifiable under the first conditions that result from reacting therespective polynucleotide with the first assay reactants and in thepresence of the target nucleic acid sequence, wherein the populationcomprises at least one individual that has the identifiable genotype andat least one individual that does not have the identifiable genotype.The method can comprise determining whether at least one individual fromthe population has an identifiable minor allele of the identifiablegenotype, under the first conditions that result from reacting therespective polynucleotide with the first assay reactants in the presenceof the target nucleic acid sequence. See U.S. Patent ApplicationPublication No. 2003/0190652 to De La Vega et al.

Other Applications and Methods

In some embodiments, high-density sequence detection system 10 can beused for a variety of biological applications, or assays, other thanPCR. In some embodiments, high-density sequence detection system 10comprising optical illumination and detection system 300 can be used inimaging microplates that fit a SBS standard footprint from low densitymicroplates, for example, 96, 384, or 1536 well microplates tohigh-density microplates, for example, 6144 or 31104 well microplate. Insome embodiments, using lower density microplates high-density sequencedetection system 10 can detect multiple, discrete events within a well,for example, for imaging fluorescently tagged antibodies binding toreceptors on the surface of a cell for high-throughput cell-basedscreening. In some embodiments, high-density sequence detection system10 is not limited to imaging only microplate 20 but can be used in theimaging of gels, blots, nitrocellulose membranes, and the like withfeatures at high-density.

In some embodiments, high-density sequence detection system 10 can imagemicroplates, nitrocellulose membranes, gels, films, blots, and the like.Detection can be, in some embodiments, for isotopic changes,chemiluminescent emissions, chemifluorescent emissions, fluorescentemissions, calorimetric changes, and time-lapse studies of any of theabove detection methods. In some embodiments, high-density sequencedetection system 10 can be used as a spectrophotometer orspectrofluorometer for samples contained in microplate 20. For example,high-density sequence detection system can be used for methods for themeasurement and/or analysis of absorbance (UV-Vis-NIR) by adding adetector to opposite side from excitation side of microplate 20; formethods for the measurement and/or analysis of fluorescence intensity;for methods for the measurement and/or analysis of fluorescencepolarization by adding at least one polarizing filter to detectionsystem 300; or for methods for the measurement and/or analysis of timeresolved fluorescence. In some embodiments of high-density sequencedetection system 10 can be modified to increase read out speed of CCDpixels. In some embodiments, high-density sequence detection system 10can be used for methods for the measurement and/or analysis ofluminescence; In some embodiments, high-density sequence detectionsystem 10 can be used for time-limited chemiluminescent reactions and insuch embodiments, high-density sequence detection system 10 can bemodified to manipulate reagents in microplate 20 to begin the reactions.

Isothermal Amplification

According to some embodiments, high-density sequence detection system 10can be used to perform various isothermal procedures in, for example,the areas of molecular diagnostics, genotyping, gene expressionmonitoring, and drug screening. Such isothermal procedures can include,for example, those useful in genetic, biochemical, and bioanalyticprocesses, such as processes for detecting a target DNA, processes fordetecting a mutation, processes for detecting a polymorphism, processesfor detecting a single base insertion or deletion, and for processes foridentifying SNPs. In some embodiments, the high-density sequencedetection system 10 can be used to perform isothermal amplificationaccording to U.S. Pat. No. 6,692,917.

In some embodiments, processes for identifying SNPs can include, forexample, assays for single-base discrimination and/or quantitativedetection of DNA or RNA sequences, for example, SNPs and mutations(single base changes, insertions or deletions in DNA and RNA molecules),from samples containing genomic DNA, total RNA, cell lysates, purifiedDNA, purified RNA, or nucleic acid amplification products, for example,PCR or RT-PCR products. Other assays that can be carried out usinghigh-density sequence detection system 10 of the present teachingsinclude the processes and methods taught in U.S. Pat. No. 6,692,917.

In some embodiments, the assays can be performed using a high-densitysequence detection system 10 wherein assay 1000 comprises reactioncomponents, including, for example, the first oligonucleotide, thedetection probe, or both the first oligonucleotide and the detectionprobe. In some embodiments, such components can be attached tomicroplate 20, directly or through a spacer and/or linker molecule,including for example, a carbon chain, a polynucleotide, biotin, or apolyglycol. In some embodiments, the assays can be performed alone or incombination with nucleic acid amplification assays, including forexample, standard or multiplex PCR.

Protein Assays

In some embodiments, high-density sequence detection system 10 can beused to detect the binding activity of primary antibody reagents asdirect labeled conjugates or indirect conjugate forms, for example,conjugate enzymes or conjugate Quantum Dots (Qdots). Cells from avariety of sources can be used including in vitro tissue culture andperipheral blood leukocytes. In some embodiments, binding events can bedetected or imaged from microplate 20, or alternatively, onnitrocellulose membranes with high-density separation channels and/orbands, for example, using a Western blot technique. In some embodiments,when using a Western blot, one protein in a mixture of any number ofproteins can be detected while also providing information about the sizeof the protein and such information can indicate how much protein hasaccumulated in cells.

Referring to an illustrative example, first proteins are separated usingSDS-polyacrylamide gel electrophoresis (SDS-PAGE) which separates theproteins by size. Nitrocellulose membrane is placed on the gel and theprotein bands are electrokinetically transported onto the nitrocellulosemembrane. This results in a nitrocellulose membrane imprinted with thesame protein bands as the gel. The nitrocellulose membrane is thenincubated with a primary antibody made by inoculating a rabbit anddiluting the antisera (from blood). The primary antibody sticks to theprotein and forms an antibody-protein complex with the protein ofinterest. The nitrocellulose membrane is then incubated with a secondaryantibody, an antibody enzyme conjugate. The secondary antibody is anantibody against the primary antibody and has the ability to stick tothe primary antibody. The conjugate enzyme can comprise a molecularflare stuck onto the antibodies so they can be visualized. The enzyme isincubated in its specific reaction mix resulting in bands wherever thereis a protein-primary antibody-secondary antibody-enzyme complex such aswherever the protein of interest is located. In some embodiments,high-density sequence detection system 10 can be used to detect a flashof light that is given off by the enzyme and, in some embodiments,detection system 300 of high-density sequence detection system 10 can becustomized for the particular conjugated labels.

By way of example in some embodiments, Green Fluorescent Protein (GFP)is extracted from Aequorea Victoria. GFP is a small protein (about 27Kd) and the DNA sequences coding for GFP can be manipulated byrecombinant DNA technology to create gene fusions between GFP and anyprotein of interest. Such DNA constructs can then be introduced intoliving cells to express the GFP fluorescent tags on the protein ofinterest. The GFP fluorescent tag can be used to localize a protein ofinterest to a specific cell type and/or subcellular localization inliving cells and organisms. In some embodiments, high-density sequencedetection system 10 optics can be modified to enable 2-40× magnificationof individual wells or a small number of wells, adding an x-y stage andadding z-axis autofocus. In some embodiments, high-density sequencedetection system 10 can be used to perform GFP-based proteinlocalization assays using microplate 20. In some embodiments, for geneexpression, the GFP DNA coding sequence can be placed behind a promoterand/or regulatory DNA sequence of interest, and introduced into cellsand this can be used to perform promoter studies in living organisms.

In some embodiments, fluorescence resonance energy transfer (FRET)assays can be used to determine the exact time and place ofcolocalization. Energy transfers from the excited fluorophore to thenearby acceptor fluorophore. In some embodiments, donor and acceptormolecules are less than 10 nm apart and the emission spectra of thedonor fluorophore overlap the excitation spectra of the acceptorfluorophore. The farther apart the molecules are, the weaker thetransfer energy. Extremely low light levels require, in someembodiments, a highly sensitive cooled CCD with high quantum efficiencyand fast readout rates. FRET images can be taken at differentwavelengths. In some embodiments, high-density sequence detection system10 can be modified to perform FRET assays in microplate 20. High-densitysequence detection system 10 optics can be modified to enablemagnification (e.g., 2-40×) of individual wells or a small number ofwells, adding an x-y stage, and adding z-axis autofocus. In someembodiments, high-density sequence detection system 10 can be used toperform FRET assays using microplate 20. In some embodiments,high-density sequence detection system 10 can produce a series of timelapse images for FRET.

Assays Using QDots as Labels

Quantum dots (QDots) are fluorescent nanoparticles made of inorganicmolecules, for example, CdSe and an emission wavelength of a QDot isdetermined by its physical size. In general, QDots have large stokesshifts, with excitation wavelengths on the order of 408 nm and emissionwavelengths starting at around 520 nm and In some embodiments, Qdots canhave greater photostability, greater spectral separation, and brighteremission relative to organic fluorescent dyes. It is possible to label,or conjugate QDots to molecules of interest for molecular biologyassays, such as antibodies. Further, mixtures of QDots can be employedto provide multiplexing capability. Some embodiments include the use ofbeads coated with different QDot nanocrystals to detect gene expressionlevels. For example, 9 μm paramagnetic beads can be coated with mixturesof QDot nanocrystals. Unique spectral codes can be created using fourdifferent fluorescent colors of QDot nanocrystals coated onto the beadsat defined ratios. Then an outer protective coat can be applied andcross-linked. In some embodiments, gene-specific oligonucleotide probesare conjugated to the bead surface and each gene-specific bead can beidentified by its unique QDot nanocrystal spectral code. Gene-specificbeads can be combined to form custom gene panels. In some embodiments,many beads of each different type are added to each well 26 with thedifferent bead types having been coated with the spectral codecorresponding to the different target DNA.

Referring to an illustrative example, total RNA is isolated from cellsor tissue and the sample can then be labeled with biotin. Unbound biotincan be separated from the biotynilated-sample complex by washing, sizeexclusion, or any of a number of other well-known processes. The cleanlyseparated biotin labeled sample can then be added to the bead mixturesin microplate 20 and allowed to hybridize to the beads. A reporter canbe created by attaching streptavidin to a fifth QDot nanocrystal label.Unattached streptavidin can be separated from the QDot labeledstreptavidin in a manner similar to that used for separating the unboundbiotin, as before. Cleanly separated streptavidin can then be added tothe mix. This fifth QDot (the reporter) provides quantitativeinformation on gene expression. The QDot nanocrystal-labeledstreptavidin can bind to the biotinylated targets. To separate anyunbound, non-specific biotin and streptavidin, another wash step, orsize exclusions step, can be added to separate them from thebiotin-streptavidin complexes (sample-biotin to bead-oligo-streptavidincomplex). Alternatively, the beads can be allowed to settle to thebottom of wells 26 of microplate 20, which is then imaged. For example,QDots have been linked to immunoglobulin G (IgG) and streptavidin tolabel the breast cancer marker Her2 on the surface of fixed and livecancer cells, to stain actin and microtubule fibers in the cytoplasm,and to detect nuclear antigens inside the nucleus. In some embodiments,each bead can be identified by reading its spectral code and canquantify the amount of target hybridized to each coded bead. In someembodiments, high-density sequence detection system 10 can be optimizedfor the excitations and emissions of QDots. In some embodiments, withthe multiplexing capabilities afforded by spectral codes, a whole genomegene expression analysis can be completed on a microplate 20.

Cellular Assays

In some embodiments, with the addition of humidity control and CO₂ tothe existing temperature control-chamber, high-density sequencedetection system 10 can accommodate live cell assays in microplate 20.In some embodiments, high-density sequence detection system 10 ismodified to comprise magnification (e.g., 2-40×) and an x-y stage. Insome embodiments, throughput can be increased by imaging more than onewell at a time, with lower resolution and/or lower magnification images.

In some embodiments, using a lower magnification and/or imageresolution, high-density sequence detection system 10 can simultaneouslyread multiple wells in real time. This can be useful, for example, foroptimizing assay conditions and determining dose response curves. Insome embodiments using microplate 20, more such assays can be run inshorter time leading to better optimizations and more accurate IC50value determinations.

In some embodiments, microplate 20 can be modified using coatings,activations, and the like to make it more amenable to a particularassay. For example, for growing and staining adherent cells, forexample, high protein binding (affinity to molecules for hydrophobic andhydrophilic domains—high binding of antibodies), and for low bindingcapacity (affinity to molecules of hydrophobic domains).

In some embodiments, high-density sequence detection system 10comprising microplate 20 can be used to analyze cell differentiationsuch as identifying morphological changes following membrane dyeincorporation; analyze cell cycle employing the detection of G1, S andG2/M phases of a cell cycle; determine mitotic index by detection usingantibodies to identify M-phase specific marker; identify cell adhesionby detecting attachment and morphology; or monitor colony formation bydetecting the enumeration of one or more colonies. In some embodiments,high-density sequence detection system 10 comprising microplate 20 canbe used to study slow ion channels by employing, for example, detectionof ion flux fluorescent DiBAC4(3) reporter. In some embodiments,high-density sequence detection system 10 comprising microplate 20 canbe used to study protein kinase by using standard antibody methods;study translocation by identifying movement of proteins between plasmamembrane, cytoplasm, and the nucleus; study fluorescent proteins such asEGFP and Reef Coral Fluorescent Protein in multiplex assays; identifyquantum dots using limited spectral overlap from distinct conjugates; orto study cell based screening such as data lactamase, adipogenesis,hybridoma, expression cloning and/or lectin binding. In someembodiments, high-density sequence detection system 10 comprisingmicroplate 20 can be used to study G-protein coupled receptors. In suchembodiments, the membrane proteins are encoded by about 20% of genes andmost organisms and are critical for cellular communication, electricaland ion balances, structural integrity of cells and their adhesions, aswell as other like functions. In some embodiments, high-density sequencedetection system 10 can be used for the analysis of DNA/RNA/proteinquantitation and purity; PicoGreen/NanoOrange and Bradford assays;analysis of ELISA and/or enzyme kinetics; analysis of drug dissolutionprofiles; analysis of caspase-3 and protease assays; analyzing CatchPoint cAMP assays; analysis of IMAP kinase assays; analysis of intrinsictryptophan fluorescence; analysis of membrane permeability assays;analysis of FluoroBlok cell migration assays; analysis of delfia assays;analysis of immunohistochemistry; analysis of tissue staining; analysisof hybridization arrays; or analysis of amino assay.

Dielectric Spectroscopy of Molecular Biology Assays

In some embodiments of high-density sequence detection system 10, anelectrically conductive circuitry can be added to microplate 20 totransform a plurality of wells 26 into resonant cavities. In someembodiments, a terminal antenna can be placed in close proximity to asample in each of the plurality of wells 26, such as a coplanarwaveguide device. Such circuitry can deliver electrical signals in theHz-GHz frequency ranges, for example in the microwave ranges, to thesamples. In some embodiments, an electrical connector can be added tomicroplate 20 in order to connect it to the generated and measuredelectrical signals from external sources, such as an Agilent vectornetwork analyzer. Such a system can be used to measure changes in thedielectric properties of the samples contained in the plurality of wells26 of microplate 20. Examples of events that cause changes in dielectricproperties, which can be detected or monitored by such a system, includemonitoring cell growth and/or death, detecting DNA hybridization,detecting protein-protein and protein-small molecule interactions,detecting protein conformational changes, detecting ion channel flux incells, and monitoring bulk properties such as pH, and saltconcentration.

Monitoring Surface Plasmon Resonance in Real-Time

In some embodiments of high-density sequence detection system 10,microplate 20 can be modified to have an electrically conductive thinlayer which can be, for example, gold, on bottom wall 36 of plurality ofwells 26. In some embodiments, surface plasmon resonance (SPR) can occurwhen polarized light incident at an angle for total internal reflectionstrikes the electrically conductive layer at the interface between mediaof different refractive index, for example, microplate material withhigh refractive index and the assay 1000 with low refractive index. Insome embodiments, an evanescent wave of electric field intensity can begenerated and interacts with (is absorbed by) free electron clouds inthe gold layer. In some embodiments, this interaction can generateelectron charge density waves called plasmons and can cause a reductionin the intensity of the reflected light. High-density sequence detectionsystem 10 can be modified to illuminate microplate 20 with incidentpolarized light covering a range of incident angles. In some embodimentswith further modifications, high-density sequence detection system 10can measure reflected light at different angles of transmission frommicroplate 20. In some embodiments, the resonance angle at which theintensity minimum occurs can be a function of the refractive index ofthe solution close to the gold layer, for example, a biological sampleflowing over the gold layer in the plurality of the wells 26 ofmicroplate 20. In some embodiments, modified high-density sequencedetection system 10 can be used to detect SPR analysis such as proteininteractions, small molecule (drug candidates) interactions with theirtargets, membrane-bound receptor interactions, DNA and RNAhybridization, interactions between whole cells and viruses, recognitionof cell surface carbohydrates and molecular interactions, such asbinding and dissociation.

Determining Presence of Specific DNA Oligonucleotide Sequences UsingBioelectronic Detection

In some embodiments, high-density array of gold electrodes can beincorporated into microplate 20. In some embodiments, capture probes andsignal probes can be designed and manufactured for a specific targetDNA. In some embodiments, capture probes can be coated onto the goldelectrodes forming a monolayer on the gold surface. In some embodiments,signal probes can be tagged with ferrocenes. In some embodiments, thetarget DNA can be amplified by PCR and when added to the monolayers onthe gold electrodes, specific target DNA can hybridize to the captureprobe. An electrochemical signal can be generated when the ampliconhybridizes to the capture probe and the ferrocene-labeled signal probe,thereby bringing a reporter molecule, ferrocene, into contact with themonolayer on the gold electrode. In some embodiments, an AC voltammogramis obtained when the specific target DNA is detected in a sample, but noelectronic signal is registered when the specific target DNA is absentfrom the sample.

Optical Planar Waveguides

In some embodiments, microplate 20 can comprise a high-density array ofplanar waveguides to selectively excite only fluorophores located at ornear the surface of the waveguide. The waveguide can be constructed bydepositing a high refractive index material onto a low refractive indexmaterial. In some embodiments, a parallel laser light beam is coupledinto the waveguiding film by a diffractive grating which is etched intothe substrate material of microplate 20. In some embodiments, the lightpropagates within the waveguiding film and creates a strong evanescentfield perpendicular to the direction of propagation into the adjacentmedium, for example, one of plurality of wells 26 in microplate 20. Insome embodiments, the field strength of the evanescent wave can decayexponentially with distance, so only fluorophores at or near the surfaceare excited. In some embodiments, selective detection of DNAhybridization, immunoaffinity reactions, and membrane receptor basedassays can be analyzed using microplate 20 comprising a high-densityarray of planar waveguides.

Microplate Applications for Localized Heating, Gradient Thermocycling

In some embodiments, microplate 20 can comprise heat generatingelectronics and such electronics can be associated with, or in proximityto, one or more of plurality of wells 26 in microplate 20. In someembodiments, temperatures in a plurality of wells 26 or subsets thereofcan be controlled to create a gradient thermocycler. In someembodiments, microplate 20 comprising heat generating electronics can beused, for example, to determine optimum assay parameters such as oligomelting point temperatures and/or can be used to improve synchronizationof thermal cycling with detection system 300 in high-density sequencedetection system 10. In some embodiments, when detection system 300 islimited to reading only a portion of microplate 20 at a time, thermalcycling reactions can be started or stopped selectively by use ofmicroplate 20 comprising heat generating electronics to correspond withoptical detection.

Portals

In some embodiments, a web-based user interface can be provided thatcomprises a web-based gene exploration system operable to provideinformation to assist a user in selecting one or both of a stock assayand a custom assay. In some embodiments, the web-based gene explorationsystem can comprise a search function operable to identify geneticmaterial based on a portion of known data. The search function canprovide one or more parameters identifying gene structure or functionfor selection by the user.

In some embodiments, systems are provided comprising a web-based userinterface configured for ordering stock assays and/or requesting customdesigned assays. Such assays can then be delivered to the user. In someembodiments, such assays are configured to detect presence or expressionof genetic material. Assays that detect the presence or expression ofgenetic material can comprise assays for detecting SNPs or for detectingexpressed genes. In some embodiments, the web-based user interface canbe configured to receive criteria related to the SNP or to the expressedtranscript for which an assay is ordered. Such methods, kits, assays,web interfaces, and the like are disclosed in U.S. Patent ApplicationPublication No. 2004/0018506 to Koehler et al.

1. A method of normalizing spectral sensitivity from an instrument, theinstrument using a microplate having a plurality of reaction chambers,the instrument having a plurality of filters, the method comprising:testing a first reference standard deposited in a first reaction chamberof the plurality of reaction chambers in the microplate, wherein thefirst reference standard is operable for outputting a first referencestandard signal in response to the testing; testing a sample aliquotdeposited in a second reaction chamber of the plurality of reactionchambers, wherein the sample aliquot includes at least a portion of apolynucleotide sample, at least one dye is deposited in the secondreaction chamber, and the at least one dye is operable for outputting adata signal representative of the polynucleotide sample in response tothe testing; detecting the first reference standard signal and the datasignal; generating a normalization bias based on analyzing the firstreference standard signal relative to a predetermined level; andnormalizing the data signal relative to the normalization bias.
 2. Themethod according to claim 1, further comprising: depositing a secondreference standard into a third reaction chamber of the plurality ofreaction chambers in the microplate, wherein the second referencestandard is operable for outputting a second reference standard signal.3. The method according to claim 2, further comprising: detecting thesecond reference standard signal, wherein the second reference standardsignal is analyzed with the first reference standard signal relative toa predetermined level to generate the normalization bias.
 4. The methodaccording to claim 1 wherein the at least one dye comprises a seriesdilution.
 5. The method according to claim 1 wherein the at least onedye comprises multiple titration points.
 6. The method according toclaim 1 wherein the at least one dye comprises dye ladders.
 7. Themethod according to claim 1 further comprising performing reversetranscription on the sample aliquot.
 8. A method of normalizing dataoutput from an instrument, the instrument using a microplate having aplurality of reaction chambers, wherein a first reaction chamber of theplurality of reaction chambers is free from a fluorescing substance, themethod comprising: depositing a sample aliquot and at least one dye intoa second of the plurality of reaction chambers, the sample aliquothaving at least a portion of the polynucleotide sample, wherein the dyeis operable for outputting a data signal in response to thepolynucleotide sample; determining a level of background correctionbased on a signal from the first reaction chamber of the plurality ofreaction chambers; generating a normalization bias based on thedetermined level of background correction; and normalizing the datasignal relative to the normalization bias.
 9. The method according toclaim 8, further comprising: depositing a non-template control in thefirst reaction chamber of the plurality of reaction chambers.
 10. Themethod according to claim 8 wherein the at least one dye comprises aseries dilution.
 11. The method according to claim 8 wherein the atleast one dye comprises multiple titration points.
 12. The methodaccording to claim 8 wherein the at least one dye comprises dye ladders.13. A method of normalizing data output from an instrument, theinstrument using a microplate having a plurality of reaction chambers,the method comprising: detecting a first control signal output from afirst control in a first reaction chamber of the plurality of reactionchambers, wherein the first reaction chamber is free of a polynucleotidesample; detecting a data signal output from at least one dye, wherein asample aliquot and the at least one dye are in a second reaction chamberof the plurality of reaction chambers, the sample aliquot including atleast a portion of the polynucleotide sample, and the at least one dyeis operable for outputting the data signal in response to thepolynucleotide sample; generating a normalization bias based onanalyzing the first control signal relative to a predetermined level;and normalizing the data signal relative to the normalization bias. 14.The method according to claim 13,wherein the first control is anon-template control.
 15. The method according to claim 13 wherein theat least one dye comprises a series dilution.
 16. The method accordingto claim 13 wherein the at least one dye comprises multiple titrationpoints.
 17. The method according to claim 13 wherein the at least onedye comprises dye ladders.